Multi-arm polymer prodrugs

ABSTRACT

Provided herein are water-soluble prodrugs. The prodrugs of the invention comprise a water-soluble polymer having three or more arms, at least three of which are covalently attached to an active agent, e.g., a small molecule. The conjugates of the invention provide an optimal balance of polymer size and structure for achieving improved drug loading, since the conjugates of the invention possess three or more active agents releasably attached to a multi-armed water soluble polymer. The prodrugs of the invention are therapeutically effective, and exhibit improved properties in-vivo when compared to unmodified parent drug.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.10/943,799, filed Sep. 17, 2004, which claims the benefit of priority toU.S. Provisional Application No. 60/503,673, filed Sep. 17, 2003, and toU.S. Provisional Application Ser. No. 60/584,308, filed Jun. 30, 2004,the contents of which are incorporated herein by reference in theirentirety.

FIELD

This invention relates to multi-arm, water-soluble polymer drugconjugates, and in particular, to polymer-based prodrugs, and to methodsfor preparing, formulating and administering compositions comprisingsuch prodrugs.

BACKGROUND

Over the years, numerous methods have been proposed for improving thedelivery of biologically active agents. Challenges associated with theformulation and delivery of pharmaceutical agents can include pooraqueous solubility of the pharmaceutical agent, toxicity, lowbioavailability, instability, and rapid in-vivo degradation, to namejust a few. Although many approaches have been devised for improving thedelivery of pharmaceutical agents, no single approach is without itsdrawbacks. For instance, commonly employed drug delivery approachesaimed at solving or at least ameliorating one or more of these problemsinclude drug encapsulation, such as in a liposome, polymer matrix, orunimolecular micelle, covalent attachment to a water-soluble polymersuch as polyethylene glycol, use of gene targeting agents, and the like.

In looking more closely at some of these approaches, liposomeencapsulation is often plagued by low efficiencies of drug loading,resulting in an oftentimes inefficient and cost ineffective process.Moreover, the release rate of the active agent in a liposomalformulation depends upon dissolution or disintegration of the liposome,or diffusion of the active agent through the liposomal layers, therebylimiting the practical availability of the active agent to thebiological system. In addition, liposomal formulations are generallyrestricted to lipid soluble drugs. Polymer matrix-based formulations cansuffer from similar shortcomings, such as the inability towell-characterize such drug delivery systems, particular those that arecross-linked, and the variable release rates associated with activeagents that must diffuse out of a hydrolytically degradable polymermatrix. In comparison, conjugation of an active agent to a polymer suchas polyethylene glycol offers a more well-defined alternative, since theconjugate itself is often although not necessarily well-characterized,particularly in the case of site-specific attachment of the polymer tothe active agent. However, protein-based compositions containingmixtures of positional isomers varying in both the site(s) and number ofpolymer chains attached to a particular protein are not uncommon. Thiscan lead to problems with reproducibly preparing such compositions.

While modification of therapeutic proteins for the purpose of improvingtheir pharmaceutical utility is perhaps one of the most commonapplications of PEGylation, PEGylation has also been used, albeit to alimited degree, to improve the bioavailability and ease of formulationof small molecule therapeutics having poor aqueous solubilities. Forinstance, water-soluble polymers such as PEG have been covalentlyattached to artilinic acid to improve its aqueous solubility (Bentley,et al., U.S. Pat. No. 6,461,603). Similarly, PEG has been covalentlyattached to triazine-based compounds such as trimelamol to improve theirsolubility in water and enhance their chemical stability (Bentley, etal., WO 02/043772). Covalent attachment of PEG to bisindolyl maleimideshas been employed to improve poor bioavailability of such compounds dueto low aqueous solubility (Bentley, et al., WO 03/037384). Prodrugs ofcamptothecin having one or two molecules of camptothecin covalentlyattached to a linear polyethylene glycol have similarly been prepared(Greenwald, et al, U.S. Pat. No. 5,880,131).

Camptothecin (often abbreviated as “CPT”) is a phytotoxic alkaloid firstisolated from the wood and bark of Camptotheca acuminata (Nyssaceae),and has been shown to exhibit antitumor activity. The compound has apentacyclic ring system with an asymmetric center in lactone ring E witha 20 S configuration. The pentacyclic ring system includes apyrrolo[3,4-b]quinoline (rings A, B and C), a conjugated pyridone (ringD), and a six-membered lactone (ring E) with a 20-hydroxyl group. Due toits insolubility in water, camptothecin was initially evaluatedclinically in the form of a water-soluble carboxylate salt having thelactone ring open to form the sodium salt. The sodium salt, althoughexhibiting much improved water solubility in comparison to camptothecinitself, produced severe toxicity and demonstrated very little in vivoanticancer activity, thus demonstrating the undesirability of thisapproach.

It was later discovered that camptothecin and many of its derivativesinhibit topoisomerase, an enzyme that is required for swiveling andrelaxation of DNA during molecular events such as replication andtranscription. Camptothecin stabilizes and forms a reversibleenzyme-camptothecin-DNA ternary complex. The formation of the cleavablecomplex specifically prevents the reunion step of the breakage/unioncycle of the topoisomerase reaction. Topoisomerase I inhibitors are alsoknown to be useful in the treatment of HIV.

In an effort to address the poor aqueous solubility associated withcamptothecin and many of its derivatives, a number of synthetic effortshave been directed to derivatizing the A-ring and/or B-ring oresterifying the 20-hydroxyl to improve water-solubility whilemaintaining cytotoxic activity. For example, topotecan(9-dimethylaminomethyl-10-hydroxy CPT) and irinotecan(7-ethyl-10[4-(1-piperidino)-1-piperidino]carbonyloxy CPT), otherwiseknown as CPT-11, are two water-soluble CPT derivatives that have shownclinically useful activity. Conjugation of certain camptothecinderivatives, such as 10-hydroxycamptothecin and 11-hydroxycamptothecin,to a linear poly(ethylene glycol) molecule via an ester linkage has beendescribed as a means to form water soluble prodrugs (Greenwald, et al.,U.S. Pat. No. 6,011,042).

The clinical effectiveness of many small molecule therapeutics, andoncolytics in particular, is limited by several factors. For instance,irinotecan and other camptothecin derivatives undergo an undesirablehydrolysis of the E-ring lactone under alkaline conditions.Additionally, administration of irinotecan causes a number of troublingside effects, including leukopenia and diarrhea. Due to its severediarrheal side-effect, the dose of irinotecan that can be administeredin its conventional, unmodified form is extremely limited, thushampering the efficacy of this drug and others of this type.

These associated side effects, when severe, can be sufficient to arrestfurther development of such drugs as promising therapeutics. Additionalchallenges facing small molecules include high clearance rates, and inthe instance of anticancer agents, minimal tumor permeation andresidence time. Approaches involving the use of polymer attachment mustbalance the size of the polymer against the molecular weight of theactive agent in order to allow therapeutically effective doses to bedelivered. Finally, the synthesis of a modified or drug-deliveryenhanced active agent must result in reasonable yields, to make any suchapproach economically attractive. Thus, there exists a need for newmethods for effectively delivering drugs, and in particular smallmolecule drugs, and even more particularly oncolytics, which can reducetheir adverse and often toxic side-effects, whilst simultaneouslyimproving their efficacy and ease of formulation. Specifically, there isa need for improved methods for delivering drugs that possess an optimalbalance of bioavailability due to reduced clearance times, bioactivity,and efficacy, coupled with reduced side-effects. The present inventionmeets those needs.

SUMMARY

In one aspect, the present invention provides water-soluble prodrugs.The prodrugs of the invention comprise a water-soluble polymer havingthree or more arms, at least three of which are covalently attached toan active agent, e.g., a small molecule. The conjugates of the inventionprovide an optimal balance of polymer size and structure for achievingimproved drug loading, since the conjugates of the invention possessthree or more active agents attached, preferably releasably, to a watersoluble polymer. In one embodiment, each of the arms of the watersoluble polymer possesses an active agent covalently attached thereto,preferably by a hydrolyzable linkage.

In one embodiment, the prodrug conjugate comprises a multi-arm polymer,i.e., having three or more arms, where the conjugate comprises thefollowing generalized structure:

R(-Q-POLY₁-X-D)_(q)  I

In structure I, R is an organic radical possessing from about 3 to about150 carbon atoms, preferably from about 3 to about 50 carbon atoms, andeven more preferably from about 3 to about 10 carbon atoms, optionallycontaining one or more heteroatoms (e.g., O, S, or N). In oneembodiment, R possesses a number of carbon atoms selected from the groupconsisting of 3, 4, 5, 6, 7, 8, 9, and 10. R may be linear or cyclic,and typically, emanating therefrom are at least 3 independent polymerarms each having at least one active agent moiety covalently attachedthereto. Looking at the above structure, “q” corresponds to the numberof polymer arms emanating from “R”.

In structure I, Q is a linker, preferably one that is hydrolyticallystable. Typically, Q contains at least one heteroatom such as O, or S,or NH, where the atom proximal to R in Q, when taken together with R,typically represents a residue of the core organic radical R.Illustrative examples are provided below. Generally, Q contains from 1to about 10 atoms, or from 1 to about 5 atoms. More particularly, Qtypically contains one of the following number of atoms: 1, 2, 3, 4, 5,6, 7, 8, 9, or 10. In a particular embodiment, Q is O, S, or —NH—C(O)—.

In structure I, POLY₁ represents a water-soluble and non-peptidicpolymer. Representative polymers include poly(alkylene glycol),poly(olefinic alcohol), poly(vinylpyrrolidone),poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate),poly(saccharide), poly(α-hydroxy acid), poly(acrylic acid), poly(vinylalcohol), polyphosphazene, polyoxazoline, poly(N-acryloylmorpholine), orcopolymers or terpolymers thereof. In a particular embodiment ofstructure I, POLY₁ is a polyethylene glycol, preferably a linearpolyethylene glycol (i.e., in each arm of the overall multi-armstructure). In yet another embodiment, POLY₁ corresponds to thestructure, —(CH₂CH₂O)_(n)—, where n ranges from about 10 to about 400,preferably from about 50 to about 350.

In structure I, X is a spacer that comprises a hydrolyzable linkage,where the hydrolyzable linkage is attached directly to the active agent,D. Typically, at least one atom of the hydrolyzable linkage is containedin the active agent, D, in its unmodified form, such that uponhydrolysis of the hydrolyzable linkage comprised within X, the activeagent, D, is released. Generally speaking, the spacer, X, has an atomlength of from about 4 atoms to about 50 atoms, or more preferably fromabout 5 atoms to about 25 atoms, or even more preferably from about 5atoms to about 20 atoms. Representative spacers have a length of fromabout 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or about 20atoms.

In yet another particular embodiment, X possesses the structure: Y-Z,where Y is a spacer fragment covalently attached to Z, a hydrolyticallydegradable linkage. In certain embodiments, Z itself may not constitutea hydrolytically degradable linkage, however, when taken together withY, or at least a portion of Y, forms a linkage that is hydrolyticallydegradable.

In yet a more particular embodiment of the spacer, X, Y has thestructure: —(CR_(x)R_(y))_(a)—K—(CR_(x)R_(y))_(b)—(CH₂CH₂O)_(c)—,wherein each R_(x) and R_(y), in each occurrence, is independently H oran organic radical selected from the group consisting of alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, and substituted aryl, a ranges from 0 to 12 (i.e., can be0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12), b ranges from 0 to 12(i.e., can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12), K isselected from —C(O)—, —C(O)NH—, —NH—C(O)—, —O—, —S—, O—C(O)—, C(O)—O—,O—C(O)—O—, O—C(O)—NH—, NH—C(O)—O—, c ranges from 0 to 25, and Z isselected from C(O)—O—, O—C(O)—O—, —O—C(O)—NH—, and NH—C(O)—O—. Theparticular structure of K and of Z will depend upon the values of eachof a, b, and c, such that none of the following linkages result in theoverall structure of spacer X, —O—O—, NH—O—, NH—NH—.

Preferably, Y comprises (CH₂)_(a)—C(O)NH—(CH₂)_(0,1)—(CH₂CH₂O)₀₋₁₀.

In yet another embodiment of the spacer, X, Y has the structure:—(CR_(x)R_(y))_(a)—K—(CR_(x)R_(y))_(b)—(CH₂CH₂NH)_(c)—, where thevariables have the values previously described. In certain instances,the presence of the short ethylene oxide or ethyl amino fragments inspacer, X, can be useful in achieving good yields during preparation ofthe prodrug conjugate, since the presence of the linker can help tocircumvent problems associated with steric hindrance, due to themulti-armed reactive polymer, the structure of the active agent, or acombination of both. Preferably, c is selected from the group consistingof 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

Preferably, R_(x) and R_(y) in each occurrence are independently H orlower alkyl. In one embodiment, R_(x) and R_(y) are in each occurrenceH. In yet another embodiment, a ranges from 0 to 5. In yet anotherembodiment, b ranges from 0 to 5. In yet another embodiment, c rangesfrom 0 to 10. In yet another embodiment, K is —C(O)—NH. Any of theembodiments described herein is meant to apply not only to generalizedstructure I, but also to extend to particular combinations ofembodiments.

In yet another embodiment, R_(x) and R_(y) in each occurrence are H, ais 1, K is —C(O)—NH, and b is 0 or 1.

Representative examples of X include —CH₂—C(O)—NH—CH₂—C(O)O— (here, Ycorresponds to —CH₂—C(O)—NH—CH₂— and Z corresponds to —C(O)—O—), and—CH₂—C(O)—NH—(CH₂CH₂O)₂—C(O)—O— (here, Y corresponds to—CH₂—C(O)—NH—(CH₂CH₂O)₂— and Z corresponds to —C(O)—O—).

Returning now to structure I, D is an active agent moiety, and q (thenumber of independent polymer arms) ranges from about 3 to about 50.Preferably, q ranges from about 3 to about 25. More preferably, q isfrom 3 to about 10, and possesses a value of 3, 4, 5, 6, 7, 8, 9, or 10.

In accordance with one embodiment of the invention, the conjugatecomprises a polymer having from about 3 to about 25 active agentmolecules covalently attached thereto. More particularly, the conjugatecomprises a water soluble polymer having 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 active agentmolecules covalently attached thereto. In a further embodiment, theconjugate of the invention has from about 3 to about 8 active agentmolecules covalently attached to the water-soluble polymer. Typically,although not necessarily, the number of polymer arms will correspond tothe number of active agents covalently attached to the water solublepolymer.

The active agent moiety, D, is an active agent comprising a functionalgroup suitable for covalent attachment to the multi-armed polymerdescribed herein to form a hydrolyzable linkage, such that uponhydrolysis, the active agent is released in its unmodified form.

Preferred active agent moieties include anticancer agents.

In one embodiment, the active agent is a small molecule. In a particularembodiment, the active agent moiety is a small molecule possessing amolecular weight of less than about 1000. In yet additional embodiments,the small molecule drug possesses a molecular weight of less than about800, or even less than about 750. In yet another embodiment, the smallmolecule drug possesses a molecular weight of less than about 500 or, insome instances, even less than about 300.

In yet another embodiment, the small molecule is an oncolytic drughaving at least one hydroxyl group.

In yet a further embodiment, D represents a camptothecin compound havingthe structure:

wherein R₁-R₅ are each independently selected from the group consistingof hydrogen; halo; acyl; alkyl (e.g., C1-C6 alkyl); substituted alkyl;alkoxy (e.g., C1-C6 alkoxy); substituted alkoxy; alkenyl; alkynyl;cycloalkyl; hydroxyl; cyano; nitro; azido; amido; hydrazine; amino;substituted amino (e.g., monoalkylamino and dialkylamino);hydroxycarbonyl; alkoxycarbonyl; alkylcarbonyloxy; alkylcarbonylamino;carbamoyloxy; arylsulfonyloxy; alkylsulfonyloxy; —C(R₇)═N—(O)_(i)—R₈wherein R₇ is H, alkyl, alkenyl, cycloalkyl, or aryl, i is 0 or 1, andR₈ is H, alkyl, alkenyl, cycloalkyl, or heterocycle; and R₉C(O)O—wherein R₉ is halogen, amino, substituted amino, heterocycle,substituted heterocycle, or R₁₀—O—(CH₂)_(m)—where m is an integer of1-10 and R₁₀ is alkyl, phenyl, substituted phenyl, cycloalkyl,substituted cycloalkyl, heterocycle, or substituted heterocycle; or

R₂ together with R₃ or R₃ together with R₄ form substituted orunsubstituted methylenedioxy, ethylenedioxy, or ethyleneoxy;

R₆ is H or OR′, wherein R′ is alkyl, alkenyl, cycloalkyl, haloalkyl, orhydroxyalkyl; and

L is the site of attachment to X.

In yet another particular embodiment, D is irinotecan.

Alternatively, D is a small molecule selected from the group consistingof platins, oxymorphone analogues, steroids, quinolones, andnucleosides.

In one embodiment, D is a platin such as cis-platin, hydroxyplatin,carboplatin, or oxaliplatin.

In yet a further embodiment, D is an oxymorphone analogue such asnaloxone, methylnaltrexone, oxymorphone, codeine, oxycodone, ormorphone.

In yet an additional embodiment, D is a steroid such as budesonide,triamcinolone, or fluticasone.

In yet another embodiment, D is a quinolone, isoquinolone orfluoroquinolone such as ciprofloxacin, moxifloxacin, or palonosetron.

In yet an additional embodiment, D is a nucleoside or nucleotide such asgemcitabine, cladribine, or fludarabine.

The multi-armed polymer prodrugs of the invention possess many uniquefeatures, particularly in the instance where the small molecule is ananticancer compound. For example, in one embodiment, provided is amulti-armed polymer prodrug, which when evaluated in a suitable animalmodel for solid tumor-type cancers and administered in a therapeuticallyeffective amount, is effective to suppress tumor growth to an extentthat is at least 1.5 times that, or even twice that observed for theunmodified anticancer agent, when evaluated over a time course of 30days. In yet another embodiment, the prodrug is effective to suppresstumor growth to the above extent or even greater when evaluated over atime course of 60 days. The small molecule employed is one known topossess anticancer properties, however, by virtue of its conjugation toa multi-armed polymer as described herein, possesses significantlyimproved efficacy and pharmacokinetics in comparison to the smallmolecule, e.g., anticancer compound, itself. Suitable solid tumor typesinclude malignant sarcomas, carcinomas and lymphomas of the breast,ovaries, colon, kidney, bile duct, lung and brain.

In another aspect, the invention encompasses reactive multi-armedpolymers suitable for preparing any of the above-described prodrugconjugates.

In another aspect, the invention encompasses a pharmaceuticalcomposition comprising a multi-arm polymer prodrug conjugate asdescribed above in combination with a pharmaceutically acceptablecarrier.

Another aspect of the invention provides a method for treating variousmedical conditions in a mammalian subject. More specifically, theinvention encompasses a method of administering to a mammalian subjectin need thereof a therapeutically effective amount of a multi-armprodrug conjugate of the invention. In one embodiment, the drug moiety,D, is an anticancer agent such as a camptothecin (e.g., irinotecan), andis effective to suppress tumor growth. In a particularly preferredembodiment, a multi-armed prodrug conjugate of the invention,particularly one where D is an anticancer agent, exhibits one or more ofthe following characteristics: (i) suppresses tumor growth to an extentgreater than that of unmodified D, (ii) demonstrates a tumor retentiontime that is increased over that of unmodified D, (iii) exhibits a rateof clearance that is reduced in comparison to that of unmodified D,and/or (iv) produces reduced adverse side effects in comparison tounmodified D.

According to yet another aspect, the invention provides a method oftreating cancer or a viral infection by administering a multi-armpolymer conjugate as described herein.

In yet another aspect, the invention provides a method of treating atopoisomerase I inhibitor-related disease in a mammalian subject byadministering a therapeutically effective amount of a multi-arm polymerprodrug to a mammalian subject in need thereof, where the small moleculeis a camptothecin type molecule.

According to yet another aspect, provided herein is a method oftargeting a solid tumor in a mammalian subject. The method includes thestep of administering a therapeutically effective amount of a multi-armpolymer prodrug of an anticancer agent known to be effective in thetreatment of solid tumors to a subject diagnosed as having one or morecancerous solid tumors. As a result of said administering, the prodrugis effective to produce an inhibition of solid tumor growth in thesubject that is increased over the inhibition of solid tumor growthresulting from administration of the anticancer agent alone.

In a further aspect, a method for preparing a multi-arm polymer prodrugconjugate of the invention is provided. In the method, a small molecule,D, is provided, where the small molecule comprises a functional group,F, suitable for forming a hydrolyzable linkage, Z. The small molecule isreacted with a bifunctional spacer, Y′, comprising each a first and asecond functional group, F1 and F2. The functional group F2 is suitablefor reaction with F, and F1 may optionally be in protected form(F1-Y′-F2). The reaction is carried out under conditions effective toform a partially modified active agent comprising a hydrolyzablelinkage, Z, resulting from reaction of F and F2, which corresponds tothe structure D-Z-Y′-F1. If necessary, the method includes the optionalstep of deprotecting F1 contained in the partially modified activeagent. The method then includes the step of reacting the partiallymodified active agent, D-Z-Y′-F1, with a multi-armed water-solublepolymer comprising the structure, R(-Q-POLY₁-F3)_(q), where R, Q, POLY₁,and Q are as previously defined, and F3 is a functional group that isreactive with F1. The reaction is carried out under conditions effectiveto promote reaction between F3 and F1 to convert Y′ to Y, to therebyform a polymer prodrug having the structure, R(-Q-POLY₁-Y-Z-D)_(q),where Y is a spacer fragment, and Z is a hydrolyzable linkage, which,upon hydrolysis, releases D.

In one embodiment of the method, a stoichiometric excess in an amountgreater than “q” moles of the partially modified active agent,D-Z-Y′-F1, is reacted with the multi-armed water-soluble,R(-Q-POLY₁-F3)_(q) to drive the reaction to completion, i.e., tocovalently attach active agent to each of the reactive polymer arms.

In yet another embodiment, where the small molecule D possessesadditional functional groups reactive with F2, the method furthercomprises the step of protecting the additional functional groups withsuitable protecting groups prior to reaction with the bifunctionalspacer. These protecting groups are then removed from the smallmolecules of the prodrug product, R(-Q-POLY₁-Y-Z-D)_(q).

According to yet another aspect of the invention, provided is yetanother method for preparing a multi-arm polymer prodrug of theinvention. The method includes the step of providing a reactivemulti-arm polymer having the structure, R(-Q-POLY₁-F3)_(q), where R, Q,POLY₁, and q are as previously described, and F3 is a reactivefunctional group. The multi-arm polymer is then reacted with abifunctional spacer, Y′, comprising each a first and a second functionalgroup, F1 and F2, wherein F1 is suitable for reaction with F3, and F1 isoptionally in protected form (F1-Y′-F2). The reaction is carried outunder conditions effective to form an intermediate multi-arm polymerresulting from reaction of F3 and F1, and having the structure,R(-Q-POLY₁-Y-F2)_(q). The method further includes the optional step ofdeprotecting F2 in the intermediate multi-arm polymer,R(-Q-POLY₁-Y-F2)_(q) if such is in protected form. The intermediatemulti-arm polymer, R(-Q-POLY₁-Y-F2)_(q), is then reacted with a smallmolecule, D, comprising a functional group, F, suitable for forming ahydrolyzable linkage, Z, upon reaction of F with F2, under conditionseffective to thereby form a prodrug having the structure,R(-Q-POLY₁-Y-Z-D)_(q), where Z is a hydrolyzable linkage, which, uponhydrolysis, releases D.

Reactive functional groups such as those described above as F1, F2 andF3, are numerous and may be selected from, for example, hydroxyl, activeester (e.g., N-hydroxysuccinimidyl ester and 1-benzotriazolyl ester),active carbonate (e.g., N-hydroxysuccinimidyl carbonate,1-benzotriazolyl carbonate, p-nitrophenyl carbonate), acid halide,acetal, aldehyde having a carbon length of 1 to 25 carbons (e.g.,acetaldehyde, propionaldehyde, and butyraldehyde), aldehyde hydrate,alkenyl, acrylate, methacrylate, acrylamide, active sulfone, amine,hydrazide, thiol, alkanoic acids having a carbon length (including thecarbonyl carbon) of 1 to about 25 carbon atoms (e.g., carboxylic acid,carboxymethyl, propanoic acid, and butanoic acid), isocyanate,isothiocyanate, maleimide, vinylsulfone, dithiopyridine, vinylpyridine,iodoacetamide, epoxide, glyoxal, and dione.

In one embodiment, the bifunctional spacer, Y′ is an amino acid orderived from an amino acid. Representative amino acids have thestructure HO—C(O)—CH(R″)—NH-Gp wherein R″ is H, C1-C6 alkyl, orsubstituted C1-C6 alkyl and Gp is an amino-protecting group. In analternative embodiment, the bifunctional spacer, Y′ possesses thestructure: —C(O)—(OCH₂CH₂)₁₋₁₀—NH-Gp.

The above methods for preparing a prodrug of the invention may includethe additional steps of purifying the intermediates and/or the finalprodrug products, for example by size exclusion chromatography or ionexchange chromatography in instances in which the compounds to bepurified contain one or more ionizable groups, such as carboxyl oramino.

These and other objects and features of the invention will become morefully apparent when read in conjunction with the following detaileddescription.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph illustrating the effect of an exemplary multi-armPEG-irinotecan conjugate on the growth of HT29 human colon tumorsimplanted in athymic nude mice in comparison to an untreated controlgroup and a group treated with irinotecan as described in detail inExample 2;

FIG. 2 is a graph illustrating the effects of a variety of doses (90mg/kg; 60 mg/kg; and 40 mg/kg) of an exemplary 20 kilodalton (20 K)multi-arm PEG irinotecan conjugate on the growth of NCl-H460 human lungtumors implanted in athymic nude mice in comparison to a control groupand a group treated with irinotecan as described in Example 6;

FIG. 3 is a graph illustrating the effects of a variety of doses (90mg/kg; 60 mg/kg; and 40 mg/kg) of an exemplary 40 kilodalton (40 K)multi-arm PEG irinotecan conjugate on the growth of NCl-H460 human lungtumors implanted in athymic nude mice in comparison to a control groupand a group treated with irinotecan as described in Example 6;

FIG. 4 is a graph illustrating the effects of a variety of doses (90mg/kg; 60 mg/kg; and 40 mg/kg) of an exemplary 20 kilodalton (20 K)multi-arm PEG-irinotecan conjugate on the growth of HT29 human colontumors implanted in athymic nude mice in comparison to an untreatedcontrol group and a group treated with irinotecan as described in detailin Example 6;

FIG. 5 is a graph illustrating the effects of a variety of doses (90mg/kg; 60 mg/kg; and 40 mg/kg) of an exemplary 40 kilodalton (40 K)multi-arm PEG-irinotecan conjugate on the growth of HT29 human colontumors implanted in athymic nude mice in comparison to an untreatedcontrol group and a group treated with irinotecan as described in detailin Example 6;

FIG. 6 is a graph illustrating the concentration in venous plasma overtime of (i) an exemplary 20 kilodalton (20 K) multi-arm PEG irinotecanconjugate, and (ii) a 40 kilodalton multi-arm PEG irinotecan conjugate,following IV administration as a single dose in athymic nude miceimplanted with either HT29 human colon tumors or NCl-H460 human lungtumors as described in Example 7.

FIG. 7 is a graph illustrating the concentration in tumor tissue overtime of (i) an exemplary 20 kilodalton (20 K) multi-arm PEG irinotecanconjugate, and (ii) a 40 kilodalton multi-arm PEG irinotecan conjugate,following IV administration as a single dose in athymic nude miceimplanted with either HT29 human colon tumors or NCl-H460 human lungtumors as described in Example 7.

FIG. 8 is a graph illustrating the concentration of PEG-SN-38 in plasmaover time following IV administration of (i) an exemplary 20 kilodalton(20 K) multi-arm PEG irinotecan conjugate, or (ii) a 40 kilodaltonmulti-arm PEG irinotecan conjugate, as a single dose in athymic nudemice implanted with either HT29 human colon tumors or NCl-H460 humanlung tumors as described in Example 7.

FIG. 9 is a graph illustrating the concentration of PEG SN-38 in tumortissue over time following IV administration of (i) an exemplary 20kilodalton (20 K) multi-arm PEG irinotecan conjugate, or (ii) a 40kilodalton multi-arm PEG irinotecan conjugate, as a single dose inathymic nude mice implanted with either HT29 human colon tumors orNCl-H460 human lung tumors as described in Example 7.

FIG. 10 is a graph illustrating the concentration of irinotecan invenous plasma over time following IV administration of (i) an exemplary20 kilodalton (20 K) multi-arm PEG irinotecan conjugate, or (ii) a 40kilodalton multi-arm PEG irinotecan conjugate, or (iii) irinotecanitself as a single dose in athymic nude mice implanted with either HT29human colon tumors or NCl-H460 human lung tumors as described in Example7.

FIG. 11 is a graph illustrating the concentration of irinotecan in tumortissue over time following IV administration of (i) an exemplary 20kilodalton (20 K) multi-arm PEG irinotecan conjugate, or (ii) a 40kilodalton multi-arm PEG irinotecan conjugate, or (iii) irinotecanitself, as a single dose in athymic nude mice implanted with either HT29human colon tumors or NCl-H460 human lung tumors as described in Example7.

FIG. 12 is a graph illustrating the concentration of SN-38 in plasmaover time following IV administration of (i) an exemplary 20 kilodalton(20 K) multi-arm PEG irinotecan conjugate, or (ii) a 40 kilodaltonmulti-arm PEG irinotecan conjugate, or (iii) irinotecan itself, as asingle dose in athymic nude mice implanted with either HT29 human colontumors or NCl-H460 human lung tumors as described in Example 7.

FIG. 13 is a graph illustrating the concentration of SN-38 in tumortissue over time following IV administration of (i) an exemplary 20kilodalton (20 K) multi-arm PEG irinotecan conjugate, or (ii) a 40kilodalton multi-arm PEG irinotecan conjugate, or (iii) irinotecanitself, as a single dose in athymic nude mice implanted with either HT29human colon tumors or NCl-H460 human lung tumors as described in Example7.

DETAILED DESCRIPTION

The present invention now will be described more fully hereinafter. Thisinvention may, however, be embodied in many different forms and shouldnot be construed as limited to the embodiments set forth herein; rather,these embodiments are provided so that this disclosure will be thoroughand complete, and will fully convey the scope of the invention to thoseskilled in the art.

DEFINITIONS

It must be noted that, as used in this specification, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to a “polymer” includesa single polymer as well as two or more of the same or differentpolymers, reference to a “conjugate” refers to a single conjugate aswell as two or more of the same or different conjugates, reference to an“excipient” includes a single excipient as well as two or more of thesame or different excipients, and the like.

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions describedbelow.

A “functional group” is a group that may be used, under normalconditions of organic synthesis, to form a covalent linkage between thestructure to which it is attached and another structure, which typicallybears a further functional group. The functional group generallyincludes multiple bond(s) and/or heteroatom(s). Preferred functionalgroups for use in the polymers of the invention are described below.

The term “reactive” refers to a functional group that reacts readily orat a practical rate under conventional conditions of organic synthesis.This is in contrast to those groups that either do not react or requirestrong catalysts or impractical reaction conditions in order to react(i.e., a “nonreactive” or “inert” group).

“Not readily reactive”, with reference to a functional group present ona molecule in a reaction mixture, indicates that the group remainslargely intact under conditions effective to produce a desired reactionin the reaction mixture.

An “activated derivative” of a carboxylic acid refers to a carboxylicacid derivative which reacts readily with nucleophiles, generally muchmore readily than the underivatized carboxylic acid. Activatedcarboxylic acids include, for example, acid halides (such as acidchlorides), anhydrides, carbonates, and esters. Such esters include, forexample, imidazolyl esters, and benzotriazole esters, and imide esters,such as N-hydroxysuccinimidyl (NHS) esters. An activated derivative maybe formed in situ by reaction of a carboxylic acid with one of variousreagents, e.g. benzotriazol-1-yloxy tripyrrolidinophosphoniumhexafluorophosphate (PyBOP), preferably used in combination with1-hydroxy benzotriazole (HOBT) or 1-hydroxy-7-azabenzotriazole (HOAT);O-7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU); or bis(2-oxo-3-oxazolidinyl)phosphinicchloride (BOP-Cl).

A “protecting group” is a moiety that prevents or blocks reaction of aparticular chemically reactive functional group in a molecule undercertain reaction conditions. The protecting group will vary dependingupon the type of chemically reactive group being protected as well asthe reaction conditions to be employed and the presence of additionalreactive or protecting groups in the molecule. Functional groups whichmay be protected include, by way of example, carboxylic acid groups,amino groups, hydroxyl groups, thiol groups, carbonyl groups and thelike. Representative protecting groups for carboxylic acids includeesters (such as a p-methoxybenzyl ester), amides and hydrazides; foramino groups, carbamates (such as tert-butoxycarbonyl) and amides; forhydroxyl groups, ethers and esters; for thiol groups, thioethers andthioesters; for carbonyl groups, acetals and ketals; and the like. Suchprotecting groups are well-known to those skilled in the art and aredescribed, for example, in T. W. Greene and G. M. Wuts, ProtectingGroups in Organic Synthesis, Third Edition, Wiley, New York, 1999, andreferences cited therein.

A functional group in “protected form” refers to a functional groupbearing a protecting group. As used herein, the term “functional group”or any synonym thereof is meant to encompass protected forms thereof.

“PEG” or “poly(ethylene glycol)” as used herein, is meant to encompassany water-soluble poly(ethylene oxide). Typically, PEGs for use in thepresent invention will comprise one of the two following structures:“—(CH₂CH₂O)_(n)—” or “—(CH₂CH₂O)_(n-1)CH₂CH₂—,” depending upon whetheror not the terminal oxygen(s) has been displaced, e.g., during asynthetic transformation. The variable (n) is 3 to 3000, and theterminal groups and architecture of the overall PEG may vary. When PEGfurther comprises a spacer as in structure I above (to be described ingreater detail below), the atoms comprising the spacer (X), whencovalently attached to a PEG segment, do not result in formation of (i)an oxygen-oxygen bond (—O—O—, a peroxide linkage), or (ii) anitrogen-oxygen bond (N—O, O—N). “PEG” means a polymer that contains amajority, that is to say, greater than 50%, of subunits that are—CH₂CH₂O—. PEGs for use in the invention include PEGs having a varietyof molecular weights, structures or geometries to be described ingreater detail below.

“Water-soluble”, in the context of a polymer of the invention or a“water-soluble polymer segment” is any segment or polymer that issoluble in water at room temperature. Typically, a water-soluble polymeror segment will transmit at least about 75%, more preferably at leastabout 95% of light, transmitted by the same solution after filtering. Ona weight basis, a water-soluble polymer or segment thereof willpreferably be at least about 35% (by weight) soluble in water, morepreferably at least about 50% (by weight) soluble in water, still morepreferably about 70% (by weight) soluble in water, and still morepreferably about 85% (by weight) soluble in water. It is most preferred,however, that the water-soluble polymer or segment is about 95% (byweight) soluble in water or completely soluble in water.

An “end-capping” or “end-capped” group is an inert group present on aterminus of a polymer such as PEG. An end-capping group is one that doesnot readily undergo chemical transformation under typical syntheticreaction conditions. An end capping group is generally an alkoxy group,—OR, where R is an organic radical comprised of 1-20 carbons and ispreferably lower alkyl (e.g., methyl, ethyl) or benzyl. “R” may besaturated or unsaturated, and includes aryl, heteroaryl, cyclo,heterocyclo, and substituted forms of any of the foregoing. Forinstance, an end capped PEG will typically comprise the structure“RO—(CH₂CH₂O)_(n)—”, where R is as defined above. Alternatively, theend-capping group can also advantageously comprise a detectable label.When the polymer has an end-capping group comprising a detectable label,the amount or location of the polymer and/or the moiety (e.g., activeagent) to which the polymer is coupled, can be determined by using asuitable detector. Such labels include, without limitation, fluorescers,chemiluminescers, moieties used in enzyme labeling, colorimetric (e.g.,dyes), metal ions, radioactive moieties, and the like.

“Non-naturally occurring” with respect to a polymer of the inventionmeans a polymer that in its entirety is not found in nature. Anon-naturally occurring polymer of the invention may however contain oneor more subunits or segments of subunits that are naturally occurring,so long as the overall polymer structure is not found in nature.

“Molecular mass” in the context of a water-soluble polymer of theinvention such as PEG, refers to the nominal average molecular mass of apolymer, typically determined by size exclusion chromatography, lightscattering techniques, or intrinsic velocity determination in1,2,4-trichlorobenzene. The polymers of the invention are typicallypolydisperse, possessing low polydispersity values of less than about1.20.

The term “linker” is used herein to refer to an atom or a collection ofatoms used to link interconnecting moieties, such as an organic radicalcore and a polymer segment, POLY₁. A linker moiety may be hydrolyticallystable or may include a physiologically hydrolyzable or enzymaticallydegradable linkage. A linker designated herein as Q is hydrolyticallystable.

The term “spacer” is used herein to refer to a collection of atoms usedto link interconnecting moieties, such as POLY₁ and the active agent, D.A spacer moiety may be hydrolytically stable or may include aphysiologically hydrolyzable or enzymatically degradable linkage. Aspacer designated herein as X comprises a hydrolyzable linkage, wherethe hydrolyzable linkage is attached directly to the active agent, D,such that upon hydrolysis, the active agent is released in its parentform.

A “hydrolyzable” bond is a relatively weak bond that reacts with water(i.e., is hydrolyzed) under physiological conditions. The tendency of abond to hydrolyze in water will depend not only on the general type oflinkage connecting two central atoms but also on the substituentsattached to these central atoms. Illustrative hydrolytically unstablelinkages include carboxylate ester, phosphate ester, anhydrides,acetals, ketals, acyloxyalkyl ether, imines, orthoesters, peptides andoligonucleotides.

An “enzymatically degradable linkage” means a linkage that is subject todegradation by one or more enzymes. Such a linkage requires the actionof one or more enzymes to effect degradation.

A “hydrolytically stable” linkage or bond refers to a chemical bond,typically a covalent bond, that is substantially stable in water, thatis to say, does not undergo hydrolysis under physiological conditions toany appreciable extent over an extended period of time. Examples ofhydrolytically stable linkages include but are not limited to thefollowing: carbon-carbon bonds (e.g., in aliphatic chains), ethers,amides, urethanes, and the like. Generally, a hydrolytically stablelinkage is one that exhibits a rate of hydrolysis of less than about1-2% per day under physiological conditions. Hydrolysis rates ofrepresentative chemical bonds can be found in most standard chemistrytextbooks.

“Multi-armed” in reference to the geometry or overall structure of apolymer refers to polymer having 3 or more polymer-containing “arms”.Thus, a multi-armed polymer may possess 3 polymer arms, 4 polymer arms,5 polymer arms, 6 polymer arms, 7 polymer arms, 8 polymer arms or more,depending upon its configuration and core structure. One particular typeof highly branched polymer is a dendritic polymer or dendrimer, that forthe purposes of the invention, is considered to possess a structuredistinct from that of a multi-armed polymer.

“Branch point” refers to a bifurcation point comprising one or moreatoms at which a polymer splits or branches from a linear structure intoone or more additional polymer arms. A multi-arm polymer may have onebranch point or multiple branch points.

A “dendrimer” is a globular, size monodisperse polymer in which allbonds emerge radially from a central focal point or core with a regularbranching pattern and with repeat units that each contribute a branchpoint. Dendrimers exhibit certain dendritic state properties such ascore encapsulation, making them unique from other types of polymers.

“Substantially” or “essentially” means nearly totally or completely, forinstance, 95% or greater of some given quantity.

“Alkyl” refers to a hydrocarbon chain, typically ranging from about 1 to20 atoms in length. Such hydrocarbon chains are preferably but notnecessarily saturated and may be branched or straight chain, althoughtypically straight chain is preferred. Exemplary alkyl groups includemethyl, ethyl, propyl, butyl, pentyl, 1-methylbutyl, 1-ethylpropyl,3-methylpentyl, and the like. As used herein, “alkyl” includescycloalkyl when three or more carbon atoms are referenced.

“Lower alkyl” refers to an alkyl group containing from 1 to 6 carbonatoms, and may be straight chain or branched, as exemplified by methyl,ethyl, n-butyl, i-butyl, t-butyl.

“Cycloalkyl” refers to a saturated or unsaturated cyclic hydrocarbonchain, including bridged, fused, or spiro cyclic compounds, preferablymade up of 3 to about 12 carbon atoms, more preferably 3 to about 8.

“Non-interfering substituents” are those groups that, when present in amolecule, are typically non-reactive with other functional groupscontained within the molecule.

The term “substituted” as in, for example, “substituted alkyl,” refersto a moiety (e.g., an alkyl group) substituted with one or morenon-interfering substituents, such as, but not limited to: C₃-C₈cycloalkyl, e.g., cyclopropyl, cyclobutyl, and the like; halo, e.g.,fluoro, chloro, bromo, and iodo; cyano; alkoxy, lower phenyl;substituted phenyl; and the like. For substitutions on a phenyl ring,the substituents may be in any orientation (i.e., ortho, meta, or para).

“Alkoxy” refers to an —O—R group, wherein R is alkyl or substitutedalkyl, preferably C₁-C₂₀ alkyl (e.g., methoxy, ethoxy, propyloxy, etc.),preferably C₁-C₇.

As used herein, “alkenyl” refers to a branched or unbranched hydrocarbongroup of 1 to 15 atoms in length, containing at least one double bond,such as ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl,octenyl, decenyl, tetradecenyl, and the like.

The term “alkynyl” as used herein refers to a branched or unbranchedhydrocarbon group of 2 to 15 atoms in length, containing at least onetriple bond, ethynyl, n-propynyl, isopropynyl, n-butynyl, isobutynyl,octynyl, decynyl, and so forth.

“Aryl” means one or more aromatic rings, each of 5 or 6 core carbonatoms. Aryl includes multiple aryl rings that may be fused, as innaphthyl or unfused, as in biphenyl. Aryl rings may also be fused orunfused with one or more cyclic hydrocarbon, heteroaryl, or heterocyclicrings. As used herein, “aryl” includes heteroaryl.

“Heteroaryl” is an aryl group containing from one to four heteroatoms,preferably N, O, or S, or a combination thereof. Heteroaryl rings mayalso be fused with one or more cyclic hydrocarbon, heterocyclic, aryl,or heteroaryl rings.

“Heterocycle” or “heterocyclic” means one or more rings of 5-12 atoms,preferably 5-7 atoms, with or without unsaturation or aromatic characterand having at least one ring atom which is not a carbon. Preferredheteroatoms include sulfur, oxygen, and nitrogen.

“Substituted heteroaryl” is heteroaryl having one or morenon-interfering groups as substituents.

“Substituted heterocycle” is a heterocycle having one or more sidechains formed from non-interfering substituents.

“Electrophile” refers to an ion, atom, or collection of atoms that maybe ionic, having an electrophilic center, i.e., a center that iselectron seeking, capable of reacting with a nucleophile.

“Nucleophile” refers to an ion or atom or collection of atoms that maybe ionic, having a nucleophilic center, i.e., a center that is seekingan electrophilic center, and capable of reacting with an electrophile.

“Active agent” as used herein includes any agent, drug, compound,composition of matter or mixture which provides some pharmacologic,often beneficial, effect that can be demonstrated in-vivo or in vitro.This includes foods, food supplements, nutrients, nutraceuticals, drugs,vaccines, antibodies, vitamins, and other beneficial agents. As usedherein, these terms further include any physiologically orpharmacologically active substance that produces a localized or systemiceffect in a patient.

“Pharmaceutically acceptable excipient” or “pharmaceutically acceptablecarrier” refers to an excipient that can be included in the compositionsof the invention and that causes no significant adverse toxicologicaleffects to the patient.

“Pharmacologically effective amount,” “physiologically effectiveamount,” and “therapeutically effective amount” are used interchangeablyherein to mean the amount of a PEG-active agent conjugate present in apharmaceutical preparation that is needed to provide a desired level ofactive agent and/or conjugate in the bloodstream or in a target tissue.The precise amount will depend upon numerous factors, e.g., theparticular active agent, the components and physical characteristics ofpharmaceutical preparation, intended patient population, patientconsiderations, and the like, and can readily be determined by oneskilled in the art, based upon the information provided herein andavailable in the relevant literature.

“Multi-functional” in the context of a polymer of the invention means apolymer having 3 or more functional groups, where the functional groupsmay be the same or different, and are typically present on the polymertermini. Multi-functional polymers of the invention will typicallycontain from about 3-100 functional groups, or from 3-50 functionalgroups, or from 3-25 functional groups, or from 3-15 functional groups,or from 3 to 10 functional groups, i.e., contains 3, 4, 5, 6, 7, 8, 9 or10 functional groups. Typically, in reference to a polymer precursorused to prepare a polymer prodrug of the invention, the polymerpossesses 3 or more polymer arms having at the terminus of each arm afunctional group suitable for coupling to an active agent moiety via ahydrolyzable linkage.

“Difunctional” or “bifunctional” as used interchangeable herein means anentity such as a polymer having two functional groups contained therein,typically at the polymer termini. When the functional groups are thesame, the entity is said to be homodifunctional or homobifunctional.When the functional groups are different, the polymer is said to beheterodifunctional or heterobifunctional

A basic or acidic reactant described herein includes neutral, charged,and any corresponding salt forms thereof.

“Polyolefinic alcohol” refers to a polymer comprising an olefin polymerbackbone, such as polyethylene, having multiple pendant hydroxyl groupsattached to the polymer backbone. An exemplary polyolefinic alcohol ispolyvinyl alcohol.

As used herein, “non-peptidic” refers to a polymer backbonesubstantially free of peptide linkages. However, the polymer may includea minor number of peptide linkages spaced along the repeat monomersubunits, such as, for example, no more than about 1 peptide linkage perabout 50 monomer units.

The term “patient,” refers to a living organism suffering from or proneto a condition that can be prevented or treated by administration of apolymer of the invention, typically but not necessarily in the form of apolymer-active agent conjugate, and includes both humans and animals.

“Optional” or “optionally” means that the subsequently describedcircumstance may or may not occur, so that the description includesinstances where the circumstance occurs and instances where it does not.

A “small molecule” may be defined broadly as an organic, inorganic, ororganometallic compound typically having a molecular weight of less thanabout 1000. Small molecules of the invention encompass oligopeptides andother biomolecules having a molecular weight of less than about 1000.

An “active agent moiety” in reference to a prodrug conjugate of theinvention, refers to the portion or residue of the unmodified parentactive agent up to the covalent linkage resulting from covalentattachment of the drug (or an activated or chemically modified formthereof) to a polymer of the invention. Upon hydrolysis of thehydrolyzable linkage between the active agent moiety and the multi-armedpolymer, the active agent per se is released.

Multi-Arm Polymer Prodrug Conjugates—Overview

As described generally above, the polymer conjugates of the inventioncomprise a multi-arm water-soluble and non-peptidic polymer covalentlyattached to at least three active agent compounds. The conjugates of theinvention are typically prodrugs, meaning that the active agent,attached to the polymer via a hydrolytically degradable linkage, isreleased over time following administration of the conjugate to asubject. Moreover, the conjugates of the invention arewell-characterized, isolable, and purifiable compositions, in comparisonto, for example, a degradable polymer-matrix having molecules of drugencapsulated therein. The conjugates of the invention exhibit higherdrug loading characteristics when compared to their linear polymer-basedcounterparts, thus lowering the total dosage weight needed to treat aparticular disease state. That is to say, the polymer scaffold of theinvention is effective to covalently attach multiple active agentmolecules thereto, thereby allowing a greater amount of therapeuticagent (i.e., active agent) to be administered per given weight ofpolymer when compared to a linear monofunctional or bifunctional polymerof about the same size but having only one or two active agent moleculesattached thereto. The polymers used in the invention are hydrophilic innature, thereby imparting hydrophilicity to the resulting conjugates,which, particularly in the case of water-insoluble active agents,facilitates their formulation into useful pharmaceutical compositions.

Typically, the total number average molecular weight of the overallmulti-arm polymer portion of a polymer conjugate of the invention isabout 1,000 daltons (Da) to about 100,000 Da, more preferably about10,000 Da to about 60,000 Da, most preferably about 15,000 to about60,000 Da. Multi-armed polymers having a number average molecular weightof about 5,000 Da, about 8,000 Da, about 10,000 Da, about 12,000 Da,about 15,000 Da, about 20,000 Da, about 25,000 Da, about 30,000 Da,about 35,000 Da, about 40,000 Da, about 45,000 Da, about 50,000 Da, andabout 60,000 Da are particularly preferred. Multi-armed polymers havinga molecular weight of 20,000 Da or greater, i.e., of about 20,000 Da, or25,000 Da, or 30,000 Da, or 40,000 Da or 50,000 Da, or 60,000 Da, areparticularly preferred for tumor-targeting applications. The actualmolecular weight of the multi-armed polymer will depend, of course, onthe number of polymer arms and the molecular weight of each polymer armin the overall multi-armed polymer.

The linkage between the multi-armed polymer portion and the active agentis preferably hydrolytically degradable for in vivo release of theparent drug molecule over time. Representative hydrolytically degradablelinkages corresponding to X in structure I include carboxylate ester,carbonate ester, phosphate ester, anhydride, acetal, ketal, acyloxyalkylether, imine, orthoester, and oligonucleotides. Esters such ascarboxylate and carbonate esters are particularly preferred linkages.The particular linkage and linkage chemistry employed will depend uponthe particular active agent, the presence of additional functionalgroups within the active agent, and the like, and can be readilydetermined by one skilled in the art based upon the guidance presentedherein.

With respect to the multi-arm prodrug conjugates of the invention, it isnot necessary for the polymer conjugate itself to exhibit biologicalactivity, since the parent drug is released upon hydrolysis. However, incertain embodiments, the polymer conjugate maintains at least ameasurable degree of activity. That is to say, in some instances, amulti-armed polymer conjugate possesses anywhere from about 1% to about100% or more of the specific activity of the unmodified parent compound.That is to say, a multi-armed polymer prodrug of the invention willpossess from about 1% to about 100% bioactivity relative to theunmodified parent active agent, prior to conjugation. Such activity maybe determined using a suitable in-vivo or in-vitro model, depending uponthe known activity of the particular parent compound. For anticancerdrugs, in vivo anticancer activity is typically evaluated by comparisonof growth rates of tumor implants in drug treated and control groups ofathymic mice using well-established animal models (See for example,Examples 2 and 6). Anticancer activity is indicated by slower tumorgrowth rates in the treated group relative to the control group (J. W.Singer, et al., Ann. N.Y. Acad. Sci., 922: 136-150, 2000). In general,certain polymer conjugates of the invention will possess a specificactivity of at least about 2%, 5%, 10%, 15%, 25%, 30%, 40%, 50%, 60%,80%, 90% or more relative to that of the unmodified parent drug whenmeasured in a suitable model.

As demonstrated in Examples 2, 6, and 7, preferred polymer prodrugconjugates of the invention exhibit enhanced properties in comparison totheir unmodified parent drug counterparts. The polymer conjugates of theinvention exhibit enhanced permeation and retention (EPR) in targettissues by passively accumulating in such tissues, to provide targeteddelivery of the drug to desired sites in the body (See Matsumara Y,Maeda H. “A NEW CONCEPT FOR MACROMOLECULAR THERAPEUTICS IN CANCERTHERAPY; MECHANISM OF TUMORITROPIC ACCUMULATION OF PROTEINS AND THEANTITUMOUR AGENT SMANCS”, Cancer Res 1986; 46:6387-92).

Additionally, the severity of the side effects associated withadministration of the polymer conjugates of the invention is preferablycomparable to, or even more preferably, is less than, the side effectsassociated with administration of the parent compound. In particular,preferred conjugates, particularly those comprising 3 or more moleculesof an anticancer agent such as irinotecan, when administered to apatient, result in reduced leukopenia and diarrhea when compared to theunmodified parent drug molecule. The severity of side effects ofanticancer agents such as camptothecin and camptothecin-like compoundscan be readily assessed (See, for example, Kado, et al., CancerChemotherapy and Pharmacology, Aug. 6, 2003). The polymer conjugates ofthe invention are believed to exhibit reduced side effects as comparedto the unconjugated parent drug, in part, due to the accumulation of theconjugate molecules in the target tissue and away from other sites oflikely toxicity. Each of these features of the prodrugs of the inventionwill now be discussed in greater detail below.

Structural Features of the Polymer Prodrug

As described above, a prodrug of the invention comprises a multi-armpolymer, i.e., having three or more arms, where the conjugate comprisesthe following generalized structure:

R(-Q-POLY₁-X-D)_(q)

Each arm of the multi-armed prodrug is independent from the other. Thatis to say, each of the “q” arms of the prodrug may be composed of adifferent Q, POLY₁, X, D and so forth. Typical of such embodiments, ageneralized structure corresponds to:R[(-Q₁-POLY_(1A)-X₁-D₁)(Q₂-POLY_(1B)-X₂-D₂)(Q₃-POLY_(1C)-X₃-D₃)] and soforth for each of the arms emanating from the central organic core.Generally, however, each arm of the multi-armed prodrug is the same.

Each of the variable components of structure I will now be described indetail.

Organic Core, “R”

In structure I, R is an organic core radical possessing from about 3 toabout 150 carbon atoms. Preferably, R contains from about 3 to about 50carbon atoms, and even more preferably, R contains from about 3 to about10 carbon atoms. That is to say, R may possess a number of carbon atomsselected from the group consisting of 3, 4, 5, 6, 7, 8, 9, and 10. Theorganic core may optionally contain one or more heteroatoms (e.g., O, S,or N), depending of course on the particular core molecule employed. Rmay be linear or cyclic, and typically, emanating therefrom are at least3 independent polymer arms, three or more of which have at least oneactive agent moiety covalently attached thereto. Looking at Structure I,“q” corresponds to the number of polymer arms emanating from “R”. Insome instances one or more of the polymer arms may not have an activeagent covalently attached thereto, but rather may have a relativelyunreactive or unreacted functional group at its terminus, resulting froma synthesis that failed to go to completion. In this instance, D isabsent and the individual structure of at least one of the polymer armsis in its precursor form (or is a derivative thereof), i.e., having atits terminus not an active agent, D, but rather an unreacted functionalgroup.

The central core organic radical, R, is derived from a molecule thatprovides a number of polymer attachment sites approximately equal to thedesired number of water soluble and non-peptidic polymer arms.Preferably, the central core molecule of the multi-arm polymer structureis the residue of a polyol, polythiol, or a polyamine bearing at leastthree hydroxyl, thiol, or amino groups available for polymer attachment.A “polyol” is a molecule comprising a plurality (greater than 2) ofavailable hydroxyl groups. A “polythiol” is a molecule that possesses aplurality (greater than 2) thiol groups. A “polyamine” is a moleculecomprising a plurality (greater than 2) of available amino groups.Depending on the desired number of polymer arms, the precursor polyol,polyamine or polythiol, (prior to covalent attachment of POLY₁) willtypically contain 3 to about 25 hydroxyl, or amino groups or orthiolgroups, respectively, preferably from 3 to about 10 hydroxyl, aminogroups or thiol groups, (i.e., 3, 4, 5, 6, 7, 8, 9, 10), mostpreferably, will contain from 3 to about 8 (e.g., 3, 4, 5, 6, 7, or 8)hydroxyl, amino groups or thiol groups suitable for covalent attachmentof POLY₁. The polyol, polyamine or polythiol may also include otherprotected or unprotected functional groups. Focusing on organic coresderived from polyols or polyamines, although the number of interveningatoms between each hydroxyl or amino group will vary, preferred coresare those having a length of from about 1 to about 20 intervening coreatoms, such as carbon atoms, between each hydroxyl or amino group,preferably from about 1 to about 5. In referring to intervening coreatoms and lengths, —CH₂—, for example, is considered as having a lengthof one intervening atom, although the methylene group itself containsthree atoms total, since the Hs are substituents on the carbon, and—CH₂CH₂—, for instance, is considered as having a length of two carbonatoms, etc. The particular polyol or polyamine precursor depends on thedesired number of polymer arms in the final conjugate. For example, apolyol or polyamine core molecule having 4 functional groups, Q, issuitable for preparing a prodrug in accordance with structure I havingfour polymer arms extending therefrom and covalently attached to activeagent.

The precursor polyol or polyamine core will typically possess astructure R—(OH)_(p) or R—(NH₂)_(p) prior to functionalization with apolymer. The value of p corresponds to the value of q in structure I,since each functional group, typically —OH or —NH₂, in the parent coreorganic molecule, if sterically accessible and reactive, is covalentlyattached to a polymer arm, POLY₁. Note that in structure I, the variable“Q”, when taken together with R, typically represents a residue of thecore organic radical as described herein. That is to say, whendescribing preferred organic core molecules, particularly by name, thecore molecules are described in their precursor form, rather than intheir radical form after removal of, for example, a proton. So, if forexample, the organic core radical is derived from pentaerythritol, theprecursor polyol possesses the structure C(CH₂OH)₄, and the organic coreradical, together with Q, corresponds to C(CH₂O—)₄, where Q is O.

Illustrative polyols that are preferred for use as the polymer coreinclude aliphatic polyols having from 1 to 10 carbon atoms and from 1 to10 hydroxyl groups, including for example, ethylene glycol, alkanediols, alkyl glycols, alkylidene alkyl diols, alkyl cycloalkane diols,1,5-decalindiol, 4,8-bis(hydroxymethyl)tricyclodecane, cycloalkylidenediols, dihydroxyalkanes, trihydroxyalkanes, and the like. Cycloaliphaticpolyols include straight chained or closed-ring sugars and sugaralcohols, such as mannitol, sorbitol, inositol, xylitol, quebrachitol,threitol, arabitol, erythritol, adonitol, dulcitol, facose, ribose,arabinose, xylose, lyxose, rhamnose, galactose, glucose, fructose,sorbose, mannose, pyranose, altrose, talose, tagitose, pyranosides,sucrose, lactose, maltose, and the like. Additional examples ofaliphatic polyols include derivatives of glyceraldehyde, glucose,ribose, mannose, galactose, and related stereoisomers. Aromatic polyolsmay also be used, such as 1,1,1-tris(4′-hydroxyphenyl) alkanes, such as1,1,1-tris(4-hydroxyphenyl)ethane, (1,3-adamantanediyl)diphenol,2,6-bis(hydroxyalkyl)cresols,2,2′alkylene-bis(6-t-butyl-4-alkylphenols),2,2′-alkylene-bis(t-butylphenols), catechol, alkylcatechols, pyrogallol,fluoroglycinol, 1,2,4-benzenetriol, resorcinol, alkylresorcinols,dialkylresorcinols, orcinol monohydrate, olivetol, hydroquinone,alkylhydroquinones, 1,1-bi-2-naphthol, phenyl hydroquinones,dihydroxynaphthalenes, 4,4′-(9-fluorenylidene)-diphenol, anthrarobin,dithranol, bis(hydroxyphenyl)methane biphenols, dialkylstilbesterols,bis(hydroxyphenyl)alkanes, bisphenol-A and derivatives thereof,meso-hexesterol, nordihydroguairetic acid, calixarenes and derivativesthereof, tannic acid, and the like. Other core polyols that may be usedinclude crown ethers, cyclodextrins, dextrins and other carbohydrates(e.g., monosaccharides, oligosaccharides, and polysaccharides, starchesand amylase).

Preferred polyols include glycerol, trimethylolpropane, reducing sugarssuch as sorbitol or pentaerythritol, and glycerol oligomers, such ashexaglycerol. A 21-arm polymer can be synthesized usinghydroxypropyl-β-cyclodextrin, which has 21 available hydroxyl groups.

Exemplary polyamines include aliphatic polyamines such as diethylenetriamine, N,N′,N″-trimethyldiethylene triamine, pentamethyl diethylenetriamine, triethylene tetramine, tetraethylene pentamine, pentaethylenehexamine, dipropylene triamine, tripropylene tetramine,bis-(3-aminopropyl)-amine, bis-(3-aminopropyl)-methylamine, andN,N-dimethyl-dipropylene-triamine. Naturally occurring polyamines thatcan be used in the present invention include putrescine, spermidine, andspermine. Numerous suitable pentamines, tetramines, oligoamines, andpentamidine analogs suitable for use in the present invention aredescribed in Bacchi et al., Antimicrobial Agents and Chemotherapy,January 2002, p. 55-61, Vol. 46, No. 1, which is incorporated byreference herein.

Provided below are illustrative structures corresponding to the organicradical portion of the conjugate, R, and the corresponding conjugate,assuming that each of the hydroxyls in the parent polyol has beentransformed to a polymer arm. Note that the organic radicals shownbelow, derived from polyols, include the oxygens, which, in the contextof structure I, for the arms that are polymer arms, are considered aspart of Q. It is not necessary that all hydroxyls in, for example, apolyol-derived organic radical, form part of a polymer arm. In theillustrative examples below, Q is shown as O, but can equally beconsidered as corresponding to S, —NH—, or —NH—C(O)—.

Linkages, Q and X.

The linkages between the organic radical, R, and the polymer segment,POLY₁, or between POLY₁ and the active agent, D, result from thereaction of various reactive groups contained within R, POLY₁, and D.The particular coupling chemistry employed will depend upon thestructure of the active agent, the potential presence of multiplefunctional groups within the active molecule, the need forprotection/deprotection steps, the chemical stability of the activeagent, and the like, and will be readily determined by one skilled inthe art based upon the guidance herein. Illustrative linking chemistryuseful for preparing the polymer conjugates of the invention can befound, for example, in Wong, S. H., (1991), “Chemistry of ProteinConjugation and Crosslinking”, CRC Press, Boca Raton, Fla. and inBrinkley, M. (1992) “A Brief Survey of Methods for Preparing ProteinConjugates with Dyes, Haptens, and Crosslinking Reagent”s, in Bioconjug.Chem., 3, 2013. As noted above, the overall linkage between themulti-armed polymer core and each drug molecule preferably comprises ahydrolytically degradable portion, such as an ester linkage, so that theactive agent is released over time from the multi-armed polymer core.

The multi-arm polymeric conjugates provided herein (as well as thecorresponding reactive polymer precursor molecules, and so forth)comprise a linker segment, Q, and a spacer segment, X. Exemplary spacersor linkers can include segments such as those independently selectedfrom the group consisting of —O—, —S—, —NH—, —C(O)—, —O—C(O)—, —C(O)—O—,—C(O)—NH—, —NH—C(O)—NH—, —O—C(O)—NH—, —C(S)—, —CH₂—, —CH₂—CH₂—,—CH₂—CH₂—CH₂—, —CH₂—CH₂—CH₂—CH₂—, —O—CH₂—, —CH₂—O—, —O—CH₂—CH₂—,—CH₂—O—CH₂—, —CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—, —CH₂—O—CH₂—CH₂—,—CH₂—CH₂—O—CH₂—, —CH₂—CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—CH₂—,—CH₂—O—CH₂—CH₂—CH₂—, —CH₂—CH₂—O—CH₂—CH₂—, —CH₂—CH₂—CH₂—O—CH₂—,—CH₂—CH₂—CH₂—CH₂—O—, —C(O)—NH—CH₂—, —C(O)—NH—CH₂—CH₂—,—CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—C(O)—NH—, —C(O)—NH—CH₂—CH₂—CH₂—,—CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—CH₂—C(O)—NH—,—C(O)—NH—CH₂—CH₂—CH₂—CH₂—, —CH₂—C(O)—NH—CH₂—CH₂—CH₂—,—CH₂—CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—CH₂—CH₂—C(O)—NH—, —C(O)—O—CH₂—,—CH₂—C(O)—O—CH₂—, —CH₂—CH₂—, C(O)—O—CH₂—, —C(O)—O—CH₂—CH₂—,—NH—C(O)—CH₂—, —CH₂—NH—C(O)—CH₂—, —CH₂—CH₂—NH—C(O)—CH₂—,—NH—C(O)—CH₂—CH₂—, —CH₂—NH—C(O)—CH₂—CH₂—, —CH₂—CH₂—NH—C(O)—CH₂—CH₂—,—C(O)—NH—CH₂—, —C(O)—NH—CH₂—CH₂—, —O—C(O)—NH—CH₂—, —O—C(O)—NH—CH₂—CH₂—,—O—C(O)—NH—CH₂—CH₂—CH₂—, —NH—CH₂—, —NH—CH₂—CH₂—, —CH₂—NH—CH₂—,—CH₂—CH₂—NH—CH₂—, —C(O)—CH₂—, —C(O)—CH₂—CH₂—, —CH₂—C(O)—CH₂—,—CH₂—CH₂—C(O)—CH₂—, —CH₂—CH₂—C(O)—CH₂—CH₂—, —CH₂—CH₂—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—CH₂—,—O—C(O)—NH—[CH₂]₀₋₆—(OCH₂CH₂)₀₋₂—, —C(O)—NH—(CH₂)₁₋₆—NH—C(O)—,—NH—C(O)—NH—(CH₂)₁₋₆—NH—C(O)—, —O—C(O)—CH₂—, —O—C(O)—CH₂—CH₂—, and—O—C(O)—CH₂—CH₂—CH₂—.

In any of the above examples, a simple cycloalkylene group, e.g. 1,3- or1,4-cyclohexylene, may replace any two, three or four carbon alkylenegroup. For purposes of the present disclosure, however, a series ofatoms is not a spacer moiety when the series of atoms is immediatelyadjacent to a water-soluble polymer segment and the series of atoms isbut another monomer, such that the proposed spacer moiety wouldrepresent a mere extension of the polymer chain. A spacer or linker asdescribed herein may also comprise a combination of any two or more ofthe above groups, in any orientation.

Referring to structure I, Q is a linker, preferably one that ishydrolytically stable. Typically, Q contains at least one heteroatomsuch as O, or S, or NH, where the atom proximal to R in Q, when takentogether with R, typically represents a residue of the core organicradical R. Generally, Q contains from 1 to about 10 atoms, or from 1 toabout 5 atoms. Q typically contains one of the following number ofatoms: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Illustrative Qs include O, S,or —NH—C(O)—.

Again in reference to structure I, X is a spacer that comprises ahydrolyzable linkage, where the hydrolyzable linkage is attacheddirectly to the active agent, D. Typically, at least one atom of thehydrolyzable linkage is contained in the active agent in its unmodifiedform, such that upon hydrolysis of the hydrolyzable linkage comprisedwithin X, the active agent, D, is released. Generally speaking, thespacer has an atom length of from about 4 atoms to about 50 atoms, ormore preferably from about 5 atoms to about 25 atoms, or even morepreferably from about 5 atoms to about 20 atoms. Typically, the spaceris of an atom length selected from the group consisting of 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20. When consideringatom chain length, only atoms contributing to the overall distance areconsidered. For example, a spacer having the structure, —CH₂—C(O)—NH—CH₂CH₂ O—CH₂ CH₂ O—C(O)—C— has a chain length of 11 atoms, sincesubstituents are not considered to contribute significantly to thelength of the spacer.

In yet another particular embodiment, X possesses the structure: Y-Z,where Y is a spacer fragment covalently attached to Z, a hydrolyticallydegradable linkage. In certain embodiments, Z itself may not constitutea hydrolytically degradable linkage, however, when taken together withY, or at least a portion of Y, forms a linkage that is hydrolyticallydegradable.

In yet a more particular embodiment of the spacer, X, Y has thestructure: —(CR_(x)R_(y))_(a)—K—(CR_(x)R_(y))_(b)—(CH₂CH₂O)_(c)—,wherein each R₁ and R₂, in each occurrence, is independently H or anorganic radical selected from the group consisting of alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl,and substituted aryl, a ranges from 0 to 12 (i.e., can be 0, 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, or 12), b ranges from 0 to 12 (i.e., can be 0, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12), K is selected from —C(O)—,—C(O)NH—, —NH—C(O)—, —O—, —S—, O—C(O)—, C(O)—O—, O—C(O)—O—, O—C(O)—NH—,NH—C(O)—O—, c ranges from 0 to 25, and Z is selected from C(O)—O—,O—C(O)—O—, —O—C(O)—NH—, and NH—C(O)—O—. The particular structure of Kand of Z will depend upon the values of each of a, b, and c, such thatnone of the following linkages result in the overall structure of spacerX: —O—O—, NH—O—, NH—NH—.

Preferably, Y comprises (—CH₂)_(a)—C(O)NH—(CH₂)_(0,1)—(CH₂CH₂O)₀₋₁₀.

In yet another embodiment of the spacer, X, Y has the structure:—(CR_(x)R_(y))_(a)—K—(CR_(x)R_(y))_(b)—(CH₂CH₂ NH)_(c)—, where thevariables have the values previously described. In certain instances,the presence of the short ethylene oxide or ethyl amino fragments inspacer, X, can be useful in achieving good yields during preparation ofthe prodrug conjugate, since the presence of the linker can help tocircumvent problems associated with steric hindrance, due to themulti-armed reactive polymer, the structure of the active agent, or acombination of both. Preferably, c is selected from the group consistingof 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

Preferably, R_(x) and R_(y) in each occurrence are independently H orlower alkyl. In one embodiment, R_(x) and R_(y) are in each occurrenceH. In yet another embodiment, “a” ranges from 0 to 5, i.e., is selectedfrom 0, 1, 2, 3, 4, or 5. In yet another embodiment, b ranges from 0 to5, i.e., is selected from 0, 1, 2, 3, 4, or 5. In yet anotherembodiment, c ranges from 0 to 10. In yet another embodiment, K is—C(O)—NH. Any of the embodiments described herein is meant to apply notonly to generalized structure I, but also extend to particularcombinations of embodiments.

In yet another embodiment, R_(x) and R_(y) in each occurrence are H, ais 1, K is —C(O)—NH, and b is 0 or 1.

Particular examples of X include —CH₂—C(O)—NH—CH₂—C(O)O— (here, Ycorresponds to —CH₂—C(O)—NH—CH₂— and Z corresponds to —C(O)—O—), and—CH₂—C(O)—NH—(CH₂CH₂O)₂—C(O)—O— (here, Y corresponds to—CH₂—C(O)—NH—(CH₂CH₂O)₂— and Z corresponds to —C(O)—O—).

The Polymer, Poly₁

In structure I, POLY₁ represents a water-soluble and non-peptidicpolymer. POLY₁ in each polymer arm of structure I is independentlyselected, although preferably, each polymer arm will comprise the samepolymer. Preferably, each of the arms (i.e., each “(-Q-POLY₁-X-D) ofstructure I is identical. Any of a variety of polymers that arenon-peptidic and water-soluble can be used to form a conjugate inaccordance with the present invention. Examples of suitable polymersinclude, but are not limited to, poly(alkylene glycols), copolymers ofethylene glycol and propylene glycol, poly(olefinic alcohol),poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide),poly(hydroxyalkylmethacrylate), poly(saccharides), poly(α-hydroxy acid),poly(acrylic acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline,poly(N-acryloylmorpholine), such as described in U.S. Pat. No.5,629,384, which is incorporated by reference herein in its entirety,and copolymers, terpolymers, and mixtures of any one or more of theabove.

Preferably, POLY₁ is a polyethylene glycol or PEG. POLY₁ can be in anyof a number of geometries or forms, including linear chains, branched,forked, etc., although preferably POLY₁ is linear (i.e., in each arm ofthe overall multi-arm structure) or forked. A preferred structure for amulti-armed polymer prodrug having a “forked” polymer configuration isas follows:

F represents a forking group, and the remaining variables are aspreviously described. Preferably, the fork point in the forking group,F, comprises or is (—CH), though it may also be a nitrogen atom (N). Inthis way, each polymer arm is forked to possess two active agentmoieties releasably covalently attached thereto, rather than one.

Illustrative forked polymers useful for preparing a multi-armed polymerof the type shown in Fig. XII are described in U.S. Pat. No. 6,362,254.

When POLY₁ is PEG, its structure typically comprises —(CH₂CH₂O)_(n)—,where n ranges from about 5 to about 400, preferably from about 10 toabout 350, or from about 20 to about 300.

In the multi-arm embodiments described here, each polymer arm, POLY₁,typically has a molecular weight corresponding to one of the following:200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000,4000, 5000, 6000, 7000, 7500, 8000, 9000, 10000, 12,000, 15000, 17,500,18,000, 19,000, 20,000 daltons or greater. Overall molecular weights forthe multi-armed polymer configurations described herein (that is to say,the molecular weight of the multi-armed polymer as a whole) generallycorrespond to one of the following: 800, 1000, 1200, 1600, 2000, 2400,2800, 3200, 3600, 4000, 6000, 8000, 12,000, 16,000, 20,000, 24,000,28,000, 30,000, 32,000, 36,000, 40,000, 48,000, 60,000 or greater.Typically, the overall molecular weight for a multi-armed polymer of theinvention ranges from about 800 to about 60,000 daltons.

Active Agent, D.

Returning now to structure I, D is an active agent moiety, and q (thenumber of independent polymer arms) ranges from about 3 to about 50.Preferably, q ranges from about 3 to about 25. More preferably, q isfrom 3 to about 10, and possesses a value of 3, 4, 5, 6, 7, 8, 9, or 10.The active agent moiety, D contains at least one functional groupsuitable for covalent attachment to the multi-armed polymer describedherein to form a hydrolyzable linkage, such that upon hydrolysis, theactive agent is released in its unmodified form.

In accordance with one embodiment of the invention, a prodrug conjugateis characterized as a polymer having from about 3 to about 25 activeagent molecules covalently attached thereto. More particularly, theconjugate is characterized as a water soluble polymer having 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or25 active agent molecules covalently attached thereto. In a furtherembodiment, the conjugate of the invention has from about 3 to about 8active agent molecules covalently attached to the water-soluble polymer.Typically, although not necessarily, the number of polymer arms willcorrespond to the number of active agents covalently attached to thewater soluble polymer.

In yet another embodiment, rather than having multiple polymer armsemanating from a central organic radical core, a conjugate of theinvention is characterized as a water-soluble polymer having pendantactive agent moieties covalently attached thereto, each preferablycovalently attached by a degradable linkage. In such an embodiment, thestructure of the polymer prodrug conjugate is described generally asPOLY₁(X-D)_(q), where and POLY₁, X, D, and q are as set forth above, andthe polymer, typically a linear polymer, possesses “q” active agentmoieties attached thereto, typically at discrete lengths along thepolymer chain, via the spacer X which contains a hydrolyzable linkage.

In a specific embodiment, the active agent moiety is a small moleculepossessing a molecular weight of less than about 1000. In yet additionalembodiments, the small molecule drug possesses a molecular weight ofless than about 800, or even less than about 750. In yet anotherembodiment, the small molecule drug possesses a molecular weight of lessthan about 500 or, in some instances, even less than about 300.

Preferred active agent moieties include anticancer agents. Particularlypreferred are oncolytics having at least one hydroxyl group.

One preferred class of active agents are the camptothecins. In oneembodiment, a camptothecin for use in the invention corresponds to thestructure:

wherein R₁-R₅ are each independently selected from the group consistingof hydrogen; halo; acyl; alkyl (e.g., C1-C6 alkyl); substituted alkyl;alkoxy (e.g., C1-C6 alkoxy); substituted alkoxy; alkenyl; alkynyl;cycloalkyl; hydroxyl; cyano; nitro; azido; amido; hydrazine; amino;substituted amino (e.g., monoalkylamino and dialkylamino);hydroxycarbonyl; alkoxycarbonyl; alkylcarbonyloxy; alkylcarbonylamino;carbamoyloxy; arylsulfonyloxy; alkylsulfonyloxy; —C(R₇)═N—(O)_(i)—R₈wherein R₇ is H, alkyl, alkenyl, cycloalkyl, or aryl, i is 0 or 1, andR₈ is H, alkyl, alkenyl, cycloalkyl, or heterocycle; and R₉C(O)O—wherein R₉ is halogen, amino, substituted amino, heterocycle,substituted heterocycle, or R₁₀—O—(CH₂)_(m)— where m is an integer of1-10 and R₁₀ is alkyl, phenyl, substituted phenyl, cycloalkyl,substituted cycloalkyl, heterocycle, or substituted heterocycle; or

R₂ together with R₃ or R₃ together with R₄ form substituted orunsubstituted methylenedioxy, ethylenedioxy, or ethyleneoxy;

R₆ is H or OR′, wherein R′ is alkyl, alkenyl, cycloalkyl, haloalkyl, orhydroxyalkyl; and

L is the site of attachment to X.

In one particular embodiment, D is irinotecan, where the H on the20-position hydroxyl is absent in the final multi-armed prodrugconjugate.

Active agents for use in the invention include hypnotics and sedatives,psychic energizers, tranquilizers, respiratory drugs, anticonvulsants,muscle relaxants, antiparkinson agents (dopamine antagnonists),analgesics, anti-inflammatories, antianxiety drugs (anxiolytics),appetite suppressants, antimigraine agents, muscle contractants,anti-infectives (antibiotics, antivirals, antifungals, vaccines)antiarthritics, antimalarials, antiemetics, anepileptics,bronchodilators, cytokines, growth factors, anti-cancer agents,antithrombotic agents, antihypertensives, cardiovascular drugs,antiarrhythmics, antioxidants, anti-asthma agents, hormonal agentsincluding contraceptives, sympathomimetics, diuretics, lipid regulatingagents, antiandrogenic agents, antiparasitics, anticoagulants,neoplastics, antineoplastics, hypoglycemics, nutritional agents andsupplements, growth supplements, antienteritis agents, vaccines,antibodies, diagnostic agents, and contrasting agents.

More particularly, the active agent may fall into one of a number ofstructural classes, including but not limited to small molecules,oligopeptides, polypeptides or protein mimetics, fragments, oranalogues, steroids, nucleotides, oligonucleotides, electrolytes, andthe like. Preferably, an active agent for use in the invention possessesa free hydroxyl, carboxyl, thio, amino group, or the like (i.e.,“handle”) suitable for covalent attachment to the polymer. Preferably,an active agent possesses at least one functional group suitable forforming a hydrolyzable linkage when reacted with a multi-armed polymerprecursor suitable for forming a prodrug conjugate of the invention.

Alternatively, the drug is modified by introduction of a suitable“handle”, preferably by conversion of one of its existing functionalgroups to a functional group suitable for formation of a hydrolyzablecovalent linkage between the multi-armed polymer and the drug. Ideally,such a modification should not adversely impact the therapeutic effector activity of the active agent to a significant degree. That is to say,any modification of an active agent to facilitate its attachment to amulti-armed polymer of the invention should result in no greater thanabout a 30% reduction of its bioactivity relative to the known parentactive agent prior to modification. More preferably, any modification ofan active agent to facilitate its attachment to a multi-armed polymer ofthe invention preferably results in a reduction of its activity relativeto the known parent active agent prior to modification of no greaterthan about 25%, 20%, 15%, 10% or 5%.

Specific examples of active agents include proteins, small moleculemimetics thereof, and active fragments (including variants) of thefollowing: asparaginase, amdoxovir (DAPD), antide, becaplermin,calcitonins, cyanovirin, denileukin diftitox, erythropoietin (EPO), EPOagonists (e.g., peptides from about 10-40 amino acids in length andcomprising a particular core sequence as described in WO 96/40749),dornase alpha, erythropoiesis stimulating protein (NESP), coagulationfactors such as Factor V, Factor VII, Factor VIIa, Factor VIII, FactorIX, Factor X, Factor XII, Factor XIII, von Willebrand factor; ceredase,cerezyme, alpha-glucosidase, collagen, cyclosporin, alpha defensins,beta defensins, exedin-4, granulocyte colony stimulating factor (GCSF),thrombopoietin (TPO), alpha-1 proteinase inhibitor, elcatonin,granulocyte macrophage colony stimulating factor (GMCSF), fibrinogen,filgrastim, growth hormones human growth hormone (hGH), growth hormonereleasing hormone (GHRH), GRO-beta, GRO-beta antibody, bone morphogenicproteins such as bone morphogenic protein-2, bone morphogenic protein-6,OP-1; acidic fibroblast growth factor, basic fibroblast growth factor,CD-40 ligand, heparin, human serum albumin, low molecular weight heparin(LMWH), interferons such as interferon alpha, interferon beta,interferon gamma, interferon omega, interferon tau, consensusinterferon; interleukins and interleukin receptors such as interleukin-1receptor, interleukin-2, interleukin-2 fusion proteins, interleukin-1receptor antagonist, interleukin-3, interleukin-4, interleukin-4receptor, interleukin-6, interleukin-8, interleukin-12, interleukin-13receptor, interleukin-17 receptor; lactoferrin and lactoferrinfragments, luteinizing hormone releasing hormone (LHRH), insulin,pro-insulin, insulin analogues (e.g., mono-acylated insulin as describedin U.S. Pat. No. 5,922,675), amylin, C-peptide, somatostatin,somatostatin analogs including ocreotide, vasopressin, folliclestimulating hormone (FSH), influenza vaccine, insulin-like growth factor(IGF), insulintropin, macrophage colony stimulating factor (M-CSF),plasminogen activators such as alteplase, urokinase, reteplase,streptokinase, pamiteplase, lanoteplase, and teneteplase; nerve growthfactor (NGF), osteoprotegerin, platelet-derived growth factor, tissuegrowth factors, transforming growth factor-1, vascular endothelialgrowth factor, leukemia inhibiting factor, keratinocyte growth factor(KGF), glial growth factor (GGF), T Cell receptors, CDmolecules/antigens, tumor necrosis factor (TNF), monocytechemoattractant protein-1, endothelial growth factors, parathyroidhormone (PTH), glucagon-like peptide, somatotropin, thymosin alpha 1,thymosin alpha 1 IIb/IIIa inhibitor, thymosin beta 10, thymosin beta 9,thymosin beta 4, alpha-1 antitrypsin, phosphodiesterase (PDE) compounds,VLA-4 (very late antigen-4), VLA-4 inhibitors, bisphosphonates,respiratory syncytial virus antibody, cystic fibrosis transmembraneregulator (CFTR) gene, deoxyreibonuclease (Dnase),bactericidal/permeability increasing protein (BPI), and anti-CMVantibody. Exemplary monoclonal antibodies include etanercept (a dimericfusion protein consisting of the extracellular ligand-binding portion ofthe human 75 kD TNF receptor linked to the Fc portion of IgG1),abciximab, afeliomomab, basiliximab, daclizumab, infliximab, ibritumomabtiuexetan, mitumomab, muromonab-CD3, iodine 131 tositumomab conjugate,olizumab, rituximab, and trastuzumab (herceptin).

Additional agents suitable include but are not limited to amifostine,amiodarone, aminocaproic acid, aminohippurate sodium, aminoglutethimide,aminolevulinic acid, aminosalicylic acid, amsacrine, anagrelide,anastrozole, asparaginase, anthracyclines, bexarotene, bicalutamide,bleomycin, buserelin, busulfan, cabergoline, capecitabine, carboplatin,carmustine, chlorambucin, cilastatin sodium, cisplatin, cladribine,clodronate, cyclophosphamide, cyproterone, cytarabine, camptothecins,13-cis retinoic acid, all trans retinoic acid; dacarbazine,dactinomycin, daunorubicin, deferoxamine, dexamethasone, diclofenac,diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estramustine,etoposide, exemestane, fexofenadine, fludarabine, fludrocortisone,fluorouracil, fluoxymesterone, flutamide, gemcitabine, epinephrine,L-Dopa, hydroxyurea, idarubicin, ifosfamide, imatinib, irinotecan,itraconazole, goserelin, letrozole, leucovorin, levamisole, lisinopril,lovothyroxine sodium, lomustine, mechlorethamine, medroxyprogesterone,megestrol, melphalan, mercaptopurine, metaraminol bitartrate,methotrexate, metoclopramide, mexiletine, mitomycin, mitotane,mitoxantrone, naloxone, nicotine, nilutamide, octreotide, oxaliplatin,pamidronate, pentostatin, pilcamycin, porfimer, prednisone,procarbazine, prochlorperazine, ondansetron, raltitrexed, sirolimus,streptozocin, tacrolimus, tamoxifen, temozolomide, teniposide,testosterone, tetrahydrocannabinol, thalidomide, thioguanine, thiotepa,topotecan, tretinoin, valrubicin, vinblastine, vincristine, vindesine,vinorelbine, dolasetron, granisetron; formoterol, fluticasone,leuprolide, midazolam, alprazolam, amphotericin B, podophyllotoxins,nucleoside antivirals, aroyl hydrazones, sumatriptan; macrolides such aserythromycin, oleandomycin, troleandomycin, roxithromycin,clarithromycin, davercin, azithromycin, flurithromycin, dirithromycin,josamycin, spiromycin, midecamycin, leucomycin, miocamycin, rokitamycin,andazithromycin, and swinolide A; fluoroquinolones such asciprofloxacin, ofloxacin, levofloxacin, trovafloxacin, alatrofloxacin,moxifloxicin, norfloxacin, enoxacin, grepafloxacin, gatifloxacin,lomefloxacin, sparfloxacin, temafloxacin, pefloxacin, amifloxacin,fleroxacin, tosufloxacin, prulifloxacin, irloxacin, pazufloxacin,clinafloxacin, and sitafloxacin; aminoglycosides such as gentamicin,netilmicin, paramecin, tobramycin, amikacin, kanamycin, neomycin, andstreptomycin, vancomycin, teicoplanin, rampolanin, mideplanin, colistin,daptomycin, gramicidin, colistimethate; polymixins such as polymixin B,capreomycin, bacitracin, penems; penicillins includingpenicillinase-sensitive agents like penicillin G, penicillin V;penicillinase-resistant agents like methicillin, oxacillin, cloxacillin,dicloxacillin, floxacillin, nafcillin; gram negative microorganismactive agents like ampicillin, amoxicillin, and hetacillin, cillin, andgalampicillin; antipseudomonal penicillins like carbenicillin,ticarcillin, azlocillin, meziocillin, and piperacillin; cephalosporinslike cefpodoxime, cefprozil, ceftbuten, ceftizoxime, ceftriaxone,cephalothin, cephapirin, cephalexin, cephradrine, cefoxitin,cefamandole, cefazolin, cephaloridine, cefaclor, cefadroxil,cephaloglycin, cefuroxime, ceforanide, cefotaxime, cefatrizine,cephacetrile, cefepime, cefixime, cefonicid, cefoperazone, cefotetan,cefinetazole, ceftazidime, loracarbef, and moxalactam, monobactams likeaztreonam; and carbapenems such as imipenem, meropenem, pentamidineisethiouate, albuterol sulfate, lidocaine, metaproterenol sulfate,beclomethasone diprepionate, triamcinolone acetamide, budesonideacetonide, fluticasone, ipratropium bromide, flunisolide, cromolynsodium, and ergotamine tartrate; taxanes such as paclitaxel; SN-38, andtyrphostines.

The above exemplary drugs are meant to encompass, where applicable,analogues, agonists, antagonists, inhibitors, isomers, polymorphs, andpharmaceutically acceptable salt forms thereof.

As described previously, one preferred class of active agents is thecamptothecins. The term “camptothecin compound” as used herein includesthe plant alkaloid 20(S)-camptothecin, as well as pharmaceuticallyactive derivatives, analogues and metabolites thereof. Examples ofcamptothecin derivatives include, but are not limited to,9-nitro-20(S)-camptothecin, 9-amino-20(S)-camptothecin,9-methyl-camptothecin, 9-chloro-camptothecin, 9-fluoro-camptothecin,7-ethyl camptothecin, 10-methyl-camptothecin, 10-chloro-camptothecin,10-bromo-camptothecin, 10-fluoro-camptothecin, 9-methoxy-camptothecin,11-fluoro-camptothecin, 7-ethyl-10-hydroxy camptothecin (SN38),10,11-methylenedioxy camptothecin, and 10,11-ethylenedioxy camptothecin,and 7-(4-methylpiperazinomethylene)-10,11-methylenedioxy camptothecin,7-ethyl-10-(4-(1-piperidino)-1-piperidino)-carbonyloxy-camptothecin,9-hydroxy-camptothecin, and 11-hydroxy-camptothecin. Particularlypreferred camptothecin compounds include camptothecin, irinotecan, andtopotecan.

Native and unsubstituted, the plant alkaloid camptothecin can beobtained by purification of the natural extract, or may be obtained fromthe Stehlin Foundation for Cancer Research (Houston, Tex.). Substitutedcamptothecins can be obtained using methods known in the literature orcan be obtained from commercial suppliers. For example,9-nitro-camptothecin may be obtained from SuperGen, Inc. (San Ramon,Calif.), and 9-amino-camptothecin may be obtained from IdecPharmaceuticals (San Diego, Calif.). Camptothecin and various analoguesand derivatives may also be obtained from standard fine chemical supplyhouses, such as Sigma Chemicals.

Preferred camptothecin compounds are illustrated below in Formula XI.

wherein R₁-R₅ are each independently selected from the group consistingof hydrogen; halo; acyl; alkyl (e.g., C1-C6 alkyl); substituted alkyl;alkoxy (e.g., C1-C6 alkoxy); substituted alkoxy; alkenyl; alkynyl;cycloalkyl; hydroxyl; cyano; nitro; azido; amido; hydrazine; amino;substituted amino (e.g., monoalkylamino and dialkylamino);hydroxycarbonyl; alkoxycarbonyl; alkylcarbonyloxy; alkylcarbonylamino;carbamoyloxy; arylsulfonyloxy; alkylsulfonyloxy; —C(R₇)═N—(O)_(i)—R₈wherein R₇ is H, alkyl, alkenyl, cycloalkyl, or aryl, i is 0 or 1, andR₈ is H, alkyl, alkenyl, cycloalkyl, or heterocycle; and R₉C(O)O—wherein R₉ is halogen, amino, substituted amino, heterocycle,substituted heterocycle, or R₁₀—O—(CH₂)_(m)— where m is an integer of1-10 and R₁₀ is alkyl, phenyl, substituted phenyl, cycloalkyl,substituted cycloalkyl, heterocycle, or substituted heterocycle; or

R₂ together with R₃ or R₃ together with R₄ form substituted orunsubstituted methylenedioxy, ethylenedioxy, or ethyleneoxy; and

R₆ is H or OR′, wherein R′ is alkyl, alkenyl, cycloalkyl, haloalkyl, orhydroxyalkyl.

Exemplary substituting groups include hydroxyl, amino, substitutedamino, halo, alkoxy, alkyl, cyano, nitro, hydroxycarbonyl,alkoxycarbonyl, alkylcarbonyloxy, alkylcarbonylamino, aryl (e.g.,phenyl), heterocycle, and glycosyl groups.

In one embodiment of the invention, the small molecule is not taxol, oris not taxane-based.

Other preferred active agents for preparing a multi-armed polymerprodrug conjugate as described herein include platins, oxymorphoneanalogues, steroids, quinolones, isoquinolones, and fluoroquinolones,and nucleosides and nucleotides. Structures of illustrative compoundsbelonging to each of the above structural classes are provided below.

Method of Forming a Multi-Armed Polymer Prodrug Conjugate

Multi-armed reactive polymers, such as those for preparing a prodrug ofthe invention can be readily prepared from commercially availablestarting materials in view of the guidance presented herein, coupledwith what is known in the art of chemical synthesis.

Hydroxyl-terminated multi-armed PEGs having either a pentaerythritolcore or a glycerol core are available from Nektar, Huntsville Ala. Suchmulti-armed PEGs can be used directly for coupling to active agentshaving, e.g., a carboxyl group in a position suitable for coupling,e.g., to provide a polymer prodrug having a hydrolyzable carboxyl esterbond. Alternatively, terminal hydroxyls present on a multi-armed polymerprecursor can be oxidized to terminal carboxyl groups, e.g., forcoupling to hydroxyls present on an active agent.

Alternatively, a multi-armed reactive polymer for preparing a prodrug ofthe invention may be synthetically prepared. For instance, any of anumber of suitable polyol core materials can be purchased from achemical supplier such as Aldrich (St. Louis, Mo.). The terminalhydroxyls of the polyol are first converted to their anionic form,using, for example, a strong base, to provide a site suitable forinitiating polymerization, followed by direct polymerization of monomersubunits, e.g., ethylene oxide, onto the core. Chain building is allowedto continue until a desired length of polymer chain is reached in eachof the arms, followed by terminating the reaction, e.g., by quenching.

In an alternative approach, an activated multi-armed polymer precursorto the prodrugs of the invention can be synthetically prepared by firstproviding a desired polyol core material, and reacting the polyol undersuitable conditions with a heterobifunctional PEG mesylate of a desiredlength, where the non-mesylate PEG terminus is optionally protected toprevent reaction with the polyol core. The resulting multi-armed polymerprecursor is then suitable for additional transformations or directcoupling to an active agent, following deprotection if necessary.

Multi-armed polymer precursors based on polyamino cores can be prepared,for example, by direct coupling to a polymer reagent activated with anacylating agent such as an NHS ester, a succinimidyl carbonate, a BTCester or the like, to provide multi-armed polymer precursors having anamide linker, Q. Alternatively, a core molecule having multiple aminogroups can be coupled with an aldehyde terminated polymer, such as aPEG, by reductive amination (using, for example, a reducing agent suchas sodium cyanoborohydride) to provide a multi-armed polymer precursorhaving an internal amine linker, Q.

Although the polymer PEG is described as a representative polymer in thesynthetic descriptions above, such approaches apply equally as well toother water-soluble polymers described herein.

The prodrugs of the invention can be formed using known chemicalcoupling techniques for covalent attachment of activated polymers, suchas an activated PEG, to a biologically active agent (See, for example,Poly(Ethylene Glycol) Chemistry and Biological Applications, AmericanChemical Society, Washington, D.C. (1997)). Selection of suitablefunctional groups, linkers, protecting groups, and the like to achieve amulti-arm polymer prodrug in accordance with the invention, will depend,in part, on the functional groups on the active agent and on themulti-armed polymer starting material and will be apparent to oneskilled in the art, based upon the contents of the present disclosure.

A multi-armed polymer of the invention suitable for coupling to anactive agent or derivatized active agent will typically have a terminalfunctional group such as the following: N-succinimidyl carbonate (seee.g., U.S. Pat. Nos. 5,281,698, 5,468,478), amine (see, e.g., Buckmannet al. Makromol. Chem. 182:1379 (1981), Zalipsky et al. Eur. Polym. J.19:1177 (1983)), hydrazide (See, e.g., Andresz et al. Makromol. Chem.179:301 (1978)), succinimidyl propionate and succinimidyl butanoate(see, e.g., Olson et al. in Poly(ethylene glycol) Chemistry & BiologicalApplications, pp 170-181, Harris & Zalipsky Eds., ACS, Washington, D.C.,1997; see also U.S. Pat. No. 5,672,662), succinimidyl succinate (See,e.g., Abuchowski et al. Cancer Biochem. Biophys. 7:175 (1984) andJoppich et al., Makromol. Chem. 180:1381 (1979), succinimidyl ester(see, e.g., U.S. Pat. No. 4,670,417), benzotriazole carbonate (see,e.g., U.S. Pat. No. 5,650,234), glycidyl ether (see, e.g., Pitha et al.Eur. J. Biochem. 94:11 (1979), Elling et al., Biotech. Appl. Biochem.13:354 (1991), oxycarbonylimidazole (see, e.g., Beauchamp, et al., Anal.Biochem. 131:25 (1983), Tondelli et al. J. Controlled Release 1:251(1985)), p-nitrophenyl carbonate (see, e.g., Veronese, et al., Appl.Biochem. Biotech., 11:141 (1985); and Sartore et al., Appl. Biochem.Biotech., 27:45 (1991)), aldehyde (see, e.g., Harris et al. J. Polym.Sci. Chem. Ed. 22:341 (1984), U.S. Pat. No. 5,824,784, U.S. Pat. No.5,252,714), maleimide (see, e.g., Goodson et al. Bio/Technology 8:343(1990), Romani et al. in Chemistry of Peptides and Proteins 2:29(1984)), and Kogan, Synthetic Comm. 22:2417 (1992)),orthopyridyl-disulfide (see, e.g., Woghiren, et al. Bioconj. Chem. 4:314(1993)), acrylol (see, e.g., Sawhney et al., Macromolecules, 26:581(1993)), vinylsulfone (see, e.g., U.S. Pat. No. 5,900,461).

In turning now to one of the preferred classes of active agents, thecamptothecins, since the 20-hydroxyl group of the camptothecin compoundis sterically hindered, a single step conjugation reaction is difficultto accomplish in significant yields. As a result, a preferred method isto react the 20-hydroxyl group with a short linker or spacer moietycarrying a functional group suitable for reaction with a multi-armpolymer. Such an approach is applicable to many small molecules,particularly those having a site of covalent attachment that isinaccessible to an incoming reactive polymer. Preferred linkers includet-BOC-glycine or other amino acids having a protected amino group and anavailable carboxylic acid group (See Zalipsky et al., “Attachment ofDrugs to Polyethylene Glycols”, Eur. Polym. J., Vol. 19, No. 12, pp.1177-1183 (1983)). The carboxylic acid group reacts readily with the20-hydroxyl group in the presence of a coupling agent (e.g.,dicyclohexylcarbodiimide (DCC)) and a base catalyst (e.g.,dimethylaminopyridine (DMAP)). Thereafter, the amino protecting group,such as t-BOC(N-tert-butoxycarbonyl), is removed by treatment with theappropriate deprotecting agent (e.g., trifluoroacetic acid (TFA) in thecase of t-BOC). The free amino group is then reacted with a multi-arm orforked polymer bearing carboxylic acid groups in the presence of acoupling agent (e.g., hydroxybenzyltriazole (HOBT)) and a base (e.g.,DMAP).

In a preferred embodiment, the spacer moiety is derived from andcomprises an amino acid and has the structure HO—C(O)—CH(R″)—NH-Gpwherein R″ is H, C1-C6 alkyl, or substituted C1-C6alkyl and Gp is aprotecting group protecting the alpha-amino group of the amino acid.Typical labile protecting groups include t-BOC and FMOC(9-fluorenylmethloxycarbonyl). t-BOC is stable at room temperature andeasily removed with dilute solutions of TFA and dichloromethane. FMOC isa base labile protecting group that is easily removed by concentratedsolutions of amines (usually 20-55% piperidine in N-methylpyrrolidone).Preferred amino acids include alanine, glycine, isoleucine, leucine,phenylalanine, and valine.

Other spacer moieties having an available carboxylic acid group or otherfunctional group reactive with a hydroxyl group and a protected aminogroup can also be used in lieu of the amino acids described above. Forexample, a spacer moiety having the structure HOOC-alkylene-NH-Gp may beemployed, where Gp is as described above and the alkylene chain is, forexample, about 1 to about 20 carbon atoms in length. Spacers comprisingshort —(CH₂CH₂O)_(c)— groups or (CH₂CH₂NH)_(c) groups are alsopreferred, where c varies from about 0 to about 25. More particularly, cpossesses a value selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,and 12.

In a particular embodiment exemplified in Example 1, conjugation isaccomplished by first reacting the camptothecin compound witht-BOC-glycine, followed by deprotection of the glycine amino group andcoupling of the glycine-modified camptothecin to a 4-arm PEG moleculecomprising a pentaerythritol core.

In an alternative approach exemplified in Example 8, a bifunctionalspacer comprising a number of —(CH₂CH₂O)— subunits is provided. Oneterminal functional group of the spacer is an acid chloride (—O—C(O)—Cl)suitable for reaction with an active agent hydroxyl group to form acarbonate ester (i.e., a hydrolyzable linkage), while the other terminalfunctional group is a protected amine. The bifunctional spacer iscoupled to irinotecan, in particular to the 20-position hydroxylthereof, in the presence of a coupling agent such as DMAP to provide apartially modified active agent. In the partially modified active agent,a hydrolyzable bond, Z, has been introduced, coupled to a spacer, Y′having a protected terminus, which upon deprotection, is suitable forreaction with an activated multi-armed polymer. The partially modifiedactive agent is then reacted with a multi-armed polymer precursor havinga reactive terminus suitable for coupling to an amine, to provide astable amide linkage as part of the overall linkage, X.

The prodrug product may be further purified. Methods of purification andisolation include precipitation followed by filtration and drying, aswell as chromatography. Suitable chromatographic methods include gelfiltration chromatography and ion exchange chromatography.

Pharmaceutical Compositions

The invention provides pharmaceutical formulations or compositions, bothfor veterinary and for human medical use, which comprise one or morepolymer prodrugs of the invention or a pharmaceutically acceptable saltthereof, with one or more pharmaceutically acceptable carriers, andoptionally any other therapeutic ingredients, stabilizers, or the like.The carrier(s) must be pharmaceutically acceptable in the sense of beingcompatible with the other ingredients of the formulation and not undulydeleterious to the recipient thereof. The compositions of the inventionmay also include polymeric excipients/additives or carriers, e.g.,polyvinylpyrrolidones, derivatized celluloses such ashydroxymethylcellulose, hydroxyethylcellulose, andhydroxypropylmethylcellulose, Ficolls (a polymeric sugar),hydroxyethylstarch (HES), dextrates (e.g., cyclodextrins, such as2-hydroxypropyl-β-cyclodextrin and sulfobutylether-β-cyclodextrin),polyethylene glycols, and pectin. The compositions may further includediluents, buffers, binders, disintegrants, thickeners, lubricants,preservatives (including antioxidants), flavoring agents, taste-maskingagents, inorganic salts (e.g., sodium chloride), antimicrobial agents(e.g., benzalkonium chloride), sweeteners, antistatic agents,surfactants (e.g., polysorbates such as “TWEEN 20” and “TWEEN 80”, andpluronics such as F68 and F88, available from BASF), sorbitan esters,lipids (e.g., phospholipids such as lecithin and otherphosphatidylcholines, phosphatidylethanolamines, fatty acids and fattyesters, steroids (e.g., cholesterol)), and chelating agents (e.g., EDTA,zinc and other such suitable cations). Other pharmaceutical excipientsand/or additives suitable for use in the compositions according to theinvention are listed in “Remington: The Science & Practice of Pharmacy”,19^(th) ed., Williams & Williams, (1995), and in the “Physician's DeskReference”, 52^(nd) ed., Medical Economics, Montvale, N.J. (1998), andin “Handbook of Pharmaceutical Excipients”, Third Ed., Ed. A. H. Kibbe,Pharmaceutical Press, 2000.

The prodrugs of the invention may be formulated in compositionsincluding those suitable for oral, rectal, topical, nasal, ophthalmic,or parenteral (including intraperitoneal, intravenous, subcutaneous, orintramuscular injection) administration. The compositions mayconveniently be presented in unit dosage form and may be prepared by anyof the methods well known in the art of pharmacy. All methods includethe step of bringing the active agent or compound (i.e., the prodrug)into association with a carrier that constitutes one or more accessoryingredients. In general, the compositions are prepared by bringing theactive compound into association with a liquid carrier to form asolution or a suspension, or alternatively, bring the active compoundinto association with formulation components suitable for forming asolid, optionally a particulate product, and then, if warranted, shapingthe product into a desired delivery form. Solid formulations of theinvention, when particulate, will typically comprise particles withsizes ranging from about 1 nanometer to about 500 microns. In general,for solid formulations intended for intravenous administration,particles will typically range from about 1 nm to about 10 microns indiameter. Particularly preferred are sterile, lyophilized compositionsthat are reconstituted in an aqueous vehicle prior to injection.

A preferred formulation is a solid formulation comprising the multi-armpolymer prodrug where the active agent, D, is irinotecan. The solidformulation comprises sorbitol and lactic acid, and is typically dilutedwith 5% dextrose injection or 0.9% sodium chloride injection prior tointravenous infusion.

The amount of polymer conjugate in the formulation will vary dependingupon the specific opioid antagonist employed, its activity in conjugatedform, the molecular weight of the conjugate, and other factors such asdosage form, target patient population, and other considerations, andwill generally be readily determined by one skilled in the art. Theamount of conjugate in the formulation will be that amount necessary todeliver a therapeutically effective amount of camptothecin compound to apatient in need thereof to achieve at least one of the therapeuticeffects associated with the camptothecin compound, e.g., treatment ofcancer. In practice, this will vary widely depending upon the particularconjugate, its activity, the severity of the condition to be treated,the patient population, the stability of the formulation, and the like.Compositions will generally contain anywhere from about 1% by weight toabout 99% by weight prodrug, typically from about 2% to about 95% byweight prodrug, and more typically from about 5% to 85% by weightprodrug, and will also depend upon the relative amounts ofexcipients/additives contained in the composition. More specifically,the composition will typically contain at least about one of thefollowing percentages of prodrug: 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%,or more by weight.

Compositions of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets, tablets,lozenges, and the like, each containing a predetermined amount of theactive agent as a powder or granules; or a suspension in an aqueousliquor or non-aqueous liquid such as a syrup, an elixir, an emulsion, adraught, and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine, with the active compound being in afree-flowing form such as a powder or granules which is optionally mixedwith a binder, disintegrant, lubricant, inert diluent, surface activeagent or dispersing agent. Molded tablets comprised with a suitablecarrier may be made by molding in a suitable machine.

A syrup may be made by adding the active compound to a concentratedaqueous solution of a sugar, for example sucrose, to which may also beadded any accessory ingredient(s). Such accessory ingredients mayinclude flavorings, suitable preservatives, an agent to retardcrystallization of the sugar, and an agent to increase the solubility ofany other ingredient, such as polyhydric alcohol, for example, glycerolor sorbitol.

Formulations suitable for parenteral administration convenientlycomprise a sterile aqueous preparation of the prodrug conjugate, whichcan be formulated to be isotonic with the blood of the recipient.

Nasal spray formulations comprise purified aqueous solutions of theactive agent with preservative agents and isotonic agents. Suchformulations are preferably adjusted to a pH and isotonic statecompatible with the nasal mucous membranes.

Formulations for rectal administration may be presented as a suppositorywith a suitable carrier such as cocoa butter, or hydrogenated fats orhydrogenated fatty carboxylic acids.

Ophthalmic formulations are prepared by a similar method to the nasalspray, except that the pH and isotonic factors are preferably adjustedto match that of the eye.

Topical formulations comprise the active compound dissolved or suspendedin one or more media such as mineral oil, petroleum, polyhydroxyalcohols or other bases used for topical formulations. The addition ofother accessory ingredients as noted above may be desirable.

Pharmaceutical formulations are also provided which are suitable foradministration as an aerosol, by inhalation. These formulations comprisea solution or suspension of the desired polymer conjugate or a saltthereof. The desired formulation may be placed in a small chamber andnebulized. Nebulization may be accomplished by compressed air or byultrasonic energy to form a plurality of liquid droplets or solidparticles comprising the conjugates or salts thereof.

Methods of Use

The multi-armed polymer prodrugs of the invention can be used to treator prevent any condition responsive to the unmodified active agent inany animal, particularly in mammals, including humans.

The prodrugs of the invention are particularly useful as anticanceragents, i.e., have been shown to be effective in significantly reducingthe growth of certain representative lung and colon cancers in in-vivostudies. In particular, the prodrugs of the invention have been shown tobe nearly five times more effective at preventing the growth of humanlung cancer tumors and human colon cancer tumors than the correspondinganticancer agent per se, when administered at comparable doses overillustrative time periods ranging from 30 to 80 days.

The multi-armed polymer prodrugs of the invention, in particular, thosewhere the small molecule drug is an anticancer agent such as acamptothecin compound as described herein or other oncolytic, are usefulin treating breast cancer, ovarian cancer, colon cancer, gastric cancer,malignant melanoma, small cell lung cancer, thyroid cancers, kidneycancer, cancer of the bile duct, brain cancer, lymphomas, leukemias,rhabdomyosarcoma, neuroblastoma, and the like. The prodrugs of theinvention are particularly effective in targeting and accumulating insolid tumors. The prodrugs are also useful in the treatment of HIV andother viruses.

Methods of treatment comprise administering to a mammal in need thereofa therapeutically effective amount of a composition or formulationcontaining a polymer prodrug of the invention.

A therapeutically effective dosage amount of any specific prodrug willvary from conjugate to conjugate, patient to patient, and will dependupon factors such as the condition of the patient, the activity of theparticular active agent employed, and the route of delivery.

For camptothecin-type active agents, dosages from about 0.5 to about 100mg camptothecin/kg body weight, preferably from about 10.0 to about 60mg/kg, are preferred. When administered conjointly with otherpharmaceutically active agents, even less of the prodrug may betherapeutically effective.

Methods of treatment also include administering a therapeuticallyeffective amount of a composition or formulation containing a multi-armpolymer prodrug of a camptothecin compound as described herein, inconjunction with a second anticancer agent. Preferably, suchcamptothecin type prodrugs are administered in combination with5-fluorouracil and folinic acid, as described in U.S. Pat. No.6,403,569.

The prodrug of the invention may be administered once or several times aday, preferably once a day or less. The duration of the treatment may beonce per day for a period of from two to three weeks and may continuefor a period of months or even years. The daily dose can be administeredeither by a single dose in the form of an individual dosage unit orseveral smaller dosage units or by multiple administration of subdivideddosages at certain intervals.

EXAMPLES

It is to be understood that while the invention has been described inconjunction with certain preferred specific embodiments thereof, theforegoing description as well as the examples that follow are intendedto illustrate and not limit the scope of the invention. Other aspects,advantages and modifications within the scope of the invention will beapparent to those skilled in the art to which the invention pertains.

All PEG reagents referred to in the appended examples are available fromNektar Therapeutics, Huntsville, Ala. All ¹HNMR data was generated by a300 or 400 MHz NMR spectrometer manufactured by Bruker.

ABBREVIATIONS

-   DCM: dichloromethane-   DCC dicyclohexylcarbodiimide-   DMAP dimethylaminopyridine-   HCl hydrochloric acid-   MeOH methanol-   CM carboxymethylene-   HOBT hydroxybenzyltriazole-   TFA trifluoroacetic acid-   RT room temperature-   SCM succinimidyl

Example 1 Synthesis of Pentaerythritolyl-4-Arm-(PEG-1-Methylene-2Oxo-Vinylamino Acetate Linked-Irinotecan)-20 K

A. Synthesis of t-Boc-Glycine-Irinotecan

In a flask, 0.1 g Irinotecan (0.1704 mmoles), 0.059 g t-Boc-Glycine(0.3408 mmoles), and 0.021 g DMAP (0.1704 mmoles) were dissolved in 13mL of anhydrous dichloromethane (DCM). To the solution was added 0.070 gDCC (0.3408 mmoles) dissolved in 2 mL of anhydrous DCM. The solution wasstirred overnight at room temperature. The solid was removed through acoarse frit, and the solution was washed with 10 mL of 0.1N HCL in aseparatory funnel. The organic phase was further washed with 10 mL ofdeionized H₂O in a separatory funnel and then dried with Na₂SO₄. Thesolvent was removed using rotary evaporation and the product was furtherdried under vacuum. ¹H NMR (DMSO): δ 0.919 (t, CH₂CH ₃), 1.34 (s,C(CH₃)₃), 3.83 (m, CH₂), 7.66 (d, aromatic H).

B. Deprotection of t-Boc-Glycine-Irinotecan

0.1 g t-Boc-Glycine-Irinotecan (0.137 mmoles) was dissolved in 7 mL ofanhydrous DCM. To the solution was added 0.53 mL trifluoroacetic acid(6.85 mmoles). The solution was stirred at room temperature for 1 hour.The solvent was removed using rotary evaporation. The crude product wasdissolved in 0.1 mL MeOH and then precipitated in 25 mL of ether. Thesuspension was stirred in an ice bath for 30 minutes. The product wascollected by filtration and dried under vacuum. ¹H NMR (DMSO): δ 0.92(t, CH₂CH ₃), 1.29 (t, CH₂CH ₃), 5.55 (s, 2H), 7.25 (s, aromatic H).

C. Covalent Attachment of a Multi-Armed Activated Polymer to GlycineIrinotecan.

0.516 g Glycine-Irinotecan (0.976 mmoles), 3.904 g 4arm-PEG(20 K)-CM(0.1952 mmoles), 0.0596 g 4-(dimethylamino)pyridine (DMAP, 0.488mmoles), and 0.0658 g 2-hydroxybenzyltriazole (HOBT, 0.488 mmoles) weredissolved in 60 mL anhydrous methylene chloride. To the resultingsolution was added 0.282 g 1,3-dicyclohexylcarbodiimide (DCC, 1.3664mmoles). The reaction mixture was stirred overnight at room temperature.The mixture was filtered through a coarse frit and the solvent wasremoved using rotary evaporation. The syrup was precipitated in 200 mLof cold isopropanol over an ice bath. The solid was filtered and thendried under vacuum. Yield: 4.08 g. ¹H NMR (DMSO): □0.909 (t, CH₂CH ₃),1.28 (m, CH₂CH ₃), 3.5 (br m, PEG), 3.92 (s, CH₂), 5.50 (s, 2H).

Example 2 Anti-Tumor Activity ofPentaerythritolyl-4-Arm-(PEG-1-Methylene-2 Oxo-Vinylamino AcetateLinked-Irinotecan)-20 K, “4-Arm-Peg-Gly-Irino-20 K” in a Colon CancerMouse Xenograft Model

Human HT29 colon tumor xenografts were subcutaneously implanted inathymic nude mice. After about two weeks of adequate tumor growth (100to 250 mg), these animals were divided into different groups of ten miceeach. One group was dosed with normal saline (control), a second groupwas dosed with 60 mg/kg of irinotecan, and the third group was dosedwith 60 mg/kg of the 4-arm PEG-GLY-Irino-20 K (dose calculated peririnotecan content). Doses were administered intravenously, with onedose administered every 4 days for a total of 3 administered doses. Themice were observed daily and the tumors were measured with caliperstwice a week. FIG. 1 shows the effect of irinotecan and PEG-irinotecantreatment on HT29 colon tumors in athymic nude mice.

As can be seen from the results depicted in FIG. 1, mice treated withboth irinotecan and 4-arm-PEG-GLY-Irino-20 K exhibited a delay in tumorgrowth (anti-tumor activity) that was significantly improved whencompared to the control. Moreover, the delay in tumor growth wassignificantly better for the 4-arm-PEG-GLY-Irino-20 K group of mice whencompared to the group of animals administered unconjugated irinotecan.

Example 3 Synthesis of Pentaerythritolyl-4-Arm-(PEG-1-Methylene-2Oxo-Vinylamino Acetate Linked-Irinotecan)-40 K, “4-Arm-Peg-Gly-Irino-40K”

4-arm-PEG-GLY-IRINO-40 K was prepared in an identical fashion to thatdescribed for the 20 K compound in Example 1, with the exception that instep C, the multi-armed activated PEG reagent employed was 4 arm-PEG(40K)-CM rather than the 20 K material.

Example 4 Synthesis of Pentaerythritolyl-4-Arm-(PEG-1-Methylene-2Oxo-Vinylamino Acetate Linked-SN-38)-20 K, “4-Arm-Peg-Gly-SN-38-20 k”

4-arm PEG-GLY-SN-38-20 K was prepared in a similar fashion to itsirinotecan counterpart as described in Example 1, with the exceptionthat the active agent employed was SN-38, an active metabolite ofcamptothecin, rather than irinotecan, where the phenolic-OH of SN-38 wasprotected with MEMCl (2-methoxyethoxymethyl chloride) during thechemical transformations, followed by deprotection with TEA to providethe desired multi-armed conjugate.

Example 5 Synthesis of Pentaerythritolyl-4-Arm-(PEG-1-Methylene-2Oxo-Vinylamino Acetate Linked-SN-38)-40 K, “4-Arm-Peg-Gly-SN-38-40 k”

4-arm PEG-GLY-SN-38-40 K was prepared in a similar fashion to the 20 Kversion described above, with the exception that the multi-armedactivated PEG reagent employed was 4 arm-PEG(40 K)-CM rather than the 20K material.

Example 6 Additional Xenograft Studies

Additional mouse xenograft studies were conducted to further examine theefficacy of exemplary multi-armed polymer conjugates of the invention.

Athymic nude mice were implanted subcutaneously with human cancer celllines (lung cancer cell line NCl-H460, and colon cancer cell line HT-29)and the tumors allowed to grow to approximately 150 mg in size. Theanimals were divided into groups of ten mice each.

Various compounds and doses were evaluated as follows: irinotecan (40,60 and 90 mg/kg); 4-arm-PEG-GLY-IRINO-20 K (40, 60, and 90 mg/kg);4-arm-PEG-GLY-IRINO-40 K ((40, 60, and 90 mg/kg); 4-arm-PEG-GLY-SN-38-20K (7.5, 15, 30 mg/kg), and PEG-GLY-SN-38-40 K (7.5, 15, 30 mg/kg). Doseswere administered intravenously, with one dose administered every 4 daysfor a total of 3 administered doses.

Tumor volume measurements were taken over a period of 60-80 days; tumorvolumes were converted to tumor weight. Body weights were also measuredover the same period to provide an indication of weight loss. Theresults are presented graphically in FIGS. 2-5.

Example 7 PK Study Colon Tumor Xenograft in Mice

A comparative single dose pharmacokinetic (PK) study of a multi-armedPEG-irinotecan versus unmodified irinotecan in nude mice was conductedto assess tumor distribution of parent and metabolite drug.

The study employed 108 nude mice, 36 mice per group, 4 animals persample point. Drug was administered intravenously as a single dose. Drugform and doses were as follows: irinotecan (40 mg/kg);4-arm-PEG-GLY-IRINO-20 K (40 mg/kg equivalents); 4-arm-PEG-GLY-IRINO-40K ((40 mg/kg equivalents). Venous plasma and tumor tissue samples weretaken at the following time points: 20 minutes, 40 minutes, and 1, 2, 4,12, 24, 48, and 72 hours, and evaluated for concentrations of thefollowing species: 4-arm-PEG-GLY-IRINO-20 K, 4-arm-PEG-GLY-IRINO-40 K,irinotecan and SN-38. The results are plotted in FIGS. 6 to 13.

As can be seen in FIGS. 6-13, based upon the rate of decline of themulti-armed PEGylated species in tumor tissue in comparison to plasma,the PEGylated species demonstrate a notable increase in tumor retentiontime when compared to unmodified parent drug.

In looking at the metabolite results, the concentrations of SN-38derived from the PEGylated compounds appear to be increasing at the endof the 72 hour period, while in contrast, SN-38 derived from irinotecanis essentially cleared in 12 hours. In sum, the tumor exposure to SN38following administration of either of the PEGylated compounds isapproximately five times greater than for irinotecan over the same 72hour sampling period. In sum, both multi-arm PEGylated compounds providean increased inhibition of tumor growth (colon and lung) for bothin-vivo tumor models investigated in comparison to unmodified drug. Morespecifically, both multi-arm PEGylated compounds demonstrated a markedsuppression of tumor growth when compared to unmodified drug in mousexenograft models, indicating the effectiveness of such compounds asanti-cancer agents. Lastly, administration of the multi-arm PEGylatedirinotecan compounds described herein appears to cause less diarrhea inrats than irinotecan itself.

Example 8 Synthesis ofPentaerythritolyl-4-Arm-(PEG-2-{2-[2-1-Hydroxy-2-Oxo-Vinyloxy)-Ethoxy]-Ethylamino}-Propen-1-OneLinked-Irinotecan)-20 K and -40 K

A. 2-(2-t-Boc-aminoethoxy)ethanol (1)

2-(2-Aminoethoxy)ethanol (10.5 g, 0.1 mol) and NaHCO₃ (12.6 g, 0.15 mol)were added to 100 mL CH₂Cl₂ and 100 mL H₂O. The solution was stirred atRT for 10 minutes, then di-tert-butyl dicarbonate (21.8 g, 0.1 mol) wasadded. The resulting solution was stirred at RT overnight, thenextracted with CH₂Cl₂ (3×100 mL). The organic phases were combined anddried over anhydrous sodium sulfate and evaporated under vacuum. Theresidue was subjected to silica gel column chromatography(CH₂Cl₂:CH₃OH=50:1˜10:1) to afford 2-(2-t-Boc-aminoethoxy)ethanol (1)(16.0 g, 78 mmol, yield 78%)

B. 2-(2-t-Boc-aminoethoxy)ethoxycarbonyl-Irinotecan (2)

2-(2-t-Boc-aminoethoxy)ethanol (1) (12.3 g, 60 mmol) and4-dimethylaminopyridine (DMAP) (14.6 g, 120 mmol) were dissolved in 200ml anhydrous CH₂Cl₂. Triphosgene (5.91 g, 20 mmol) was added to thesolution while stirring at room temperature. After 20 minutes, thesolution was added to a solution of irinotecan (6.0 g, 10.2 mmol) andDMAP (12.2 g, 100 mmol) in anhydrous CH₂Cl₂ (200 mL). The reaction wasstirred at RT for 2 hrs, then washed with HCl solution (pH=3, 2 L) toremove DMAP. The organic phases were combined and dried over anhydroussodium sulfate. The dried solution was evaporated under vacuum andsubjected to silica gel column chromatography (CH₂Cl₂:CH₃OH=40:1˜10:1)to afford 2-(2-t-Boc-aminoethoxy)ethoxycarbonyl-irinotecan (2) (4.9 g,6.0 mmol, yield 59%).

C. 2-(2-aminoethoxy)ethoxycarbonyl-irinotecan TFA salt (3)

2-(2-t-Boc-aminoethoxy)ethoxycarbonyl-irinotecan (2) (4.7 g, 5.75 mmol)was dissolved in 60 mL CH₂Cl₂, and trifluoroacetic acid (TFA) (20 mL)was added at RT. The reaction solution was stirred for 2 hours. Thesolvents were removed under vacuum and the residue was added to ethylether and filtered to give a yellow solid as product 3 (4.3 g, yield90%).

D. 4-arm-PEG_(20k)-carbonate-inotecan (4)

4-arm-PEG_(20k)-SCM (16.0 g) was dissolved in 200 mL CH₂Cl₂.2-(2-aminoethoxy)ethoxycarbonyl-irinotecan TFA salt (3) (2.85 g, 3.44mmol) was dissolved in 12 mL DMF and treated with 0.6 mL TEA, then addedto a solution of 4-arm-PEG_(20k)-SCM. The reaction was stirred at RT for12 hrs then precipitated in Et₂O to yield a solid product, which wasdissolved in 500 mL IPA at 50° C. The solution was cooled to RT and theresulting precipitate collected by filtration to give4-arm-PEG_(20k)-glycine-irinotecan (4) (16.2 g, drug content 7.5% basedon HPLC analysis). Yield: 60%.

E. 4-arm-PEG_(40k)-carbonate-irinotecan (5)

4-arm-PEG_(40k)-SCM (32.0 g) was dissolved in 400 mL CH₂Cl₂.2-(2-aminoethoxy)ethoxycarbonyl-irinotecan TFA salt (3) (2.85 g, 3.44mmol) was dissolved in 12 mL DMF and treated with 0.6 mL TEA, then addedto the solution of 4-arm-PEG_(40k)-SCM. The reaction was stirred at RTfor 12 hrs and then precipitated in Et₂O to get solid product, which wasdissolved in 1000 mL isopropyl alcohol (IPA) at 50° C. The solution wascooled to RT and the precipitate collected by filtration to gave4-arm-PEG_(40k)-glycine-irinotecan (4) (g, drug content 3.7% based onHPLC analysis). Yield: 59%.

Many modifications and other embodiments of the invention will come tomind to one skilled in the art to which this invention pertains havingthe benefit of the teachings presented in the foregoing description.Therefore, it is to be understood that the invention is not to belimited to the specific embodiments disclosed and that modifications andother embodiments are intended to be included within the scope of theappended claims. Although specific terms are employed herein, they areused in a generic and descriptive sense only and not for purposes oflimitation.

1. A polymer conjugate comprising a linear water-soluble andnon-peptidic polymer scaffold comprising from 3 to about 50 pendentactive agent moieties, each active agent moiety being covalentlyattached to the scaffold via a spacer comprising a hydrolyzable linkage,wherein: (i) the spacer comprises an amino acid, (ii) said scaffold hasa molecular weight ranging from about 1,000 daltons to about 100,000daltons, and (iii) said active agent moiety is a camptothecin compound.2. The conjugate of claim 1, wherein said amino acid is glycine.
 3. Theconjugate of claim 1, wherein said spacer has an atom length of frombetween about 5 and 25 atoms.
 4. The conjugate of claim 1, wherein saidhydrolyzable linkage is a carboxylate ester.
 5. The conjugate of claim4, wherein said camptothecin compound is released from said conjugatefollowing administration via hydrolysis of the ester linkage.
 6. Theconjugate of claim 1, wherein said scaffold comprises molecules of apolyol.
 7. The conjugate of claim 6, wherein said polyol is acyclodextrin.
 8. The conjugate of claim 1, wherein said scaffold is alinear water-soluble and non-peptidic copolymer.
 9. The conjugate ofclaim 8, wherein said copolymer is a copolymer of a polyethylene glycoland a poly(saccharide).
 10. The conjugate of claim 9, wherein saidpoly(saccharide) is a cyclodextrin.
 11. The conjugate of claim 1,wherein said scaffold has a molecular weight ranging from about greaterthan about 60,000 daltons to about 100,000 daltons.
 12. The conjugate ofclaim 1, which when evaluated in a suitable animal model for solidtumor-type cancers and administered in a therapeutically effectiveamount, is effective to suppress tumor growth to an extent that is atleast twice that observed for camptothecin when evaluated over a timecourse of 30 days.
 13. A conjugate having the following structure:POLY₁(X-D)_(q) wherein: POLY₁ is a linear water-soluble and non-peptidiccopolymer; D is a camptothecin compound; X is a spacer comprising anamino acid and a hydrolyzable linkage, said camptothecin compound isreleased, and (q) is from 3 to about
 50. 14. The conjugate of claim 13,wherein said amino acid is glycine.
 15. The conjugate of claim 13,wherein said hydrolyzable linkage is a carboxylate ester.
 16. Theconjugate of claim 15, wherein said camptothecin compound is releasedfrom said conjugate following administration via hydrolysis of the esterlinkage.
 17. The conjugate of claim 13, wherein said copolymer is acopolymer of a polyethylene glycol and a poly(saccharide).
 18. Theconjugate of claim 17, wherein said poly(saccharide) is a cyclodextrin.19. The conjugate of claim 13, wherein said spacer X has a structure Y-Zwhere Y is a spacer fragment covalently attached to Z, a hydrolyzablelinkage.
 20. The conjugate of claim 19, wherein Z is a carboxylateester.
 21. The conjugate of claim 20, wherein Y has the structure—(CR_(x)R_(y))_(a)—K—(CR_(x)R_(y))_(b)—(CH₂CH₂O)_(c)—, wherein eachR_(x) and R_(y), in each occurrence, is independently H or an organicradical selected from the group consisting of alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, andsubstituted aryl, a ranges from 0 to 12, b ranges from 0 to 12, c rangesfrom 0 to 25, K is selected from —C(O)—, —C(O)NH—, —NH—C(O)—, —O—, —S—,O—C(O)—, C(O)—O—, O—C(O)—O—, O—C(O)—NH—, NH—C(O)—O—.
 22. The conjugateof claim 13, which when evaluated in a suitable animal model for solidtumor-type cancers and administered in a therapeutically effectiveamount, is effective to suppress tumor growth to an extent that is atleast twice that observed for camptothecin when evaluated over a timecourse of 30 days.